Physrev 00052 2017

Physiol Rev 99: 1079 –1151, 2019
Published January 23, 2019; doi:10.1152/physrev.00052.2017
THE ROLE OF VOLTAGE-GATED SODIUM

CHANNELS IN PAIN SIGNALING
David L. Bennett, X Alex J. Clark, Jianying Huang, Stephen G. Waxman, and
Sulayman D. Dib-Hajj
Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom; Department of
Neurology and Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New
Haven, Connecticut; and Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare System,
West Haven, Connecticut
Bennett DL, Clark AJ, Huang J, Waxman SG, Dib-Hajj SD. The Role of Voltage-
L
Gated Sodium Channels in Pain Signaling. Physiol Rev 99: 1079 –1151, 2019. Pub-
lished January 23, 2019; doi:10.1152/physrev.00052.2017.—Acute pain signaling
has a key protective role and is highly evolutionarily conserved. Chronic pain, however,
is maladaptive, occurring as a consequence of injury and disease, and is associated
with sensitization of the somatosensory nervous system. Primary sensory neurons are involved
in both of these processes, and the recent advances in understanding sensory transduction
and human genetics are the focus of this review. Voltage-gated sodium channels (VGSCs) are
important determinants of sensory neuron excitability: they are essential for the initial trans-
duction of sensory stimuli, the electrogenesis of the action potential, and neurotransmitter
release from sensory neuron terminals. Nav1.1, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 are all
expressed by adult sensory neurons. The biophysical characteristics of these channels, as well
as their unique expression patterns within subtypes of sensory neurons, define their functional
role in pain signaling. Changes in the expression of VGSCs, as well as posttranslational
modifications, contribute to the sensitization of sensory neurons in chronic pain states. Fur-
thermore, gene variants in Nav1.7, Nav1.8, and Nav1.9 have now been linked to human
Mendelian pain disorders and more recently to common pain disorders such as small-fiber
neuropathy. Chronic pain affects one in five of the general population. Given the poor efficacy of
current analgesics, the selective expression of particular VGSCs in sensory neurons makes these
attractive targets for drug discovery. The increasing availability of gene sequencing, combined with
structural modeling and electrophysiological analysis of gene variants, also provides the opportunity to
better target existing therapies in a personalized manner.
I. INTRODUCTION 1079 complex array of ligand-gated ion channels and G protein-

II. PAIN AND NaV1.3 1084 coupled receptors to transduce these different stimuli. In
III. PAIN AND NaV1.7 1088 addition, these neurons express unique repertoires of volt-
IV. PAIN AND NaV1.8 1109 age-gated sodium channels (VGSCs). These are key deter-
V. PAIN AND NaV1.9 1120 minants of excitability integrating the generator potential
VI. PAIN AND NaV1.1 AND 1.6 1135 within terminals and initiating the all-or-none action poten-
VII. FINAL CONCLUSIONS 1136 tial, the propagation of action potentials to the central ner-
vous system (CNS), and finally neurotransmitter release.
Nociceptors synapse in the dorsal horn of the spinal cord
I. INTRODUCTION
where information is subject to complex modulation, both
from local circuits and from higher brain centers (480).
Nociception, as defined by Sherrington (426), is the detec-
Information is then relayed to brain stem and thalamus and
tion of actual or threatened tissue injury; the specialized
finally cortical networks (456). Sufficient activation of the
neural apparatus fulfilling this role is highly evolutionarily
conserved, providing a key protective role to the organism. nociceptive system will give rise to the perception of pain,
Nociceptors are specialized primary sensory neurons inner- an unpleasant sensory experience with both sensory dis-
vating target tissue, such as skin and muscle, which detect criminative and affective components.
injurious stimuli, whether this be extremes of temperature,
mechanical (such as high pressure), or chemicals (which VGSCs are important at every level of the somatosensory
may be endogenous mediators, such as low pH or environ- nervous system. In this review, we will principally concen-
mental irritants) (132). Each of these neurons expresses a trate on the role they have in sensory neurons, given the
0031-9333/19 Copyright © 2019 the American Physiological Society 1079

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BENNETT ET AL.
specific expression pattern in these neurons and the recently importance of voltage-dependent activation of the sodium
described human Mendelian pain disorders, which high- current (due to movement of Na⫹ into the axon) in the
light the role of these neurons. Injury can result in maladap- generation of the action potential. Subsequent studies dem-
tive plasticity within the somatosensory system, leading to onstrated that such current was carried by transmembrane
persistent pain, which is disabling and for which treatment VGSCs. These consist of a pore-forming ␣-subunit (of ~260
remains inadequate (164). Therapeutic targeting of VGSCs kDa), as well as associated ␤-subunits of smaller size
is, therefore, an important theme, both for existing analge- (30 – 40 kDa). The family of related ␣-subunits consists of
sic agents and importantly for potential new analgesics. We 10 members, 9 of which (Nav 1.1–1.9) are voltage gated
will focus on the VGSCs expressed in sensory neurons in and one further non-voltage-gated member, Nax, which is
relation to their expression, biophysical properties, and po- involved in salt sensing. The ␣-subunits of VGSCs were first
tential role in persistent pain. cloned in the 1980s (336, 337) and are composed of ~2,000
amino acids arranged in four domains (DI–DIV); each do-
A. Structure and Function of Voltage-Gated main consists of six transmembrane segments (S1–S6) (FIG-
Sodium Channels URE 1, A AND B). The S1–S4 transmembrane segments act as
a voltage sensor, and a number of positively charged amino
It was the seminal work of Hodgkin and Huxley (221–224) acids (usually arginine’s) on S4 are important for such volt-
in studying the squid giant axon, which demonstrated the age sensing. S5 and S6 form the pore. The loops between S5
FIGURE 1. Voltage-gated sodium channel structure. A: the ␣-subunit is a long polypeptide that folds into four
homologous domains (DI–DIV), each with six transmembrane spanning regions (S1–S6). S1–S4 comprises the
voltage-sensing domain (VSD), with S5 and S6 comprising the pore-forming domain (PFD). S4 (depicted in a
darker shade) characteristically contains positively charged arginine and lysine residues. The inactivation gate
is found on the intracellular linker between DIII and DIV and is composed of an IFMT (isoleucine, phenylalanine,
methionine, threonine) motif. B: structural elements of voltage-gated sodium channels from Arcobacter
butzleri (NavAb). One subunit is highlighted (transmembrane segments S1–S6); the nearest VSD has been
removed for clarity. C: architecture of NavAb pore module; pore volume is shown in gray. Important structural
elements of the pore are colored: S5 (purple), P-helix (green), selectivity filter sequence (yellow), P2-helix (red),
and S6 (purple). The pore (P) helices stabilize cations in the central cavity, and a second pore helix (P2) forms
an extracellular funnel in NavAb. D: electrostatic potential colored from ⫺10 to 10kT (red to blue). [B, C, and
D are adapted with permission from Payandeh et al. (353).]
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SODIUM CHANNELS AND PAIN
and S6 from each domain form the outer vestibule of the pseudo-unipolar in morphology. The peripheral terminals
channel, which houses the ion selectivity filter. The central of these neurons innervate the skin, muscle, or visceral tis-
pore is, therefore, made up of eight ␣-helices, surrounded by sues, and central terminals project to the dorsal horn of the
the four voltage sensors arranged in a symmetrical fashion spinal cord. Sensory neurons are very heterogeneous, dem-
around the pore (FIGURE 1, C AND D). onstrating high levels of functional specialization. They can
be most simply classified in terms of cell size, conduction
The crystal structure of a bacterial and eukaryotic VGSC velocity (207, 208), and stimulus-response function (155).
has recently been determined (FIGURE 1B) (352, 353, 425, Such classifications are increasingly now being correlated
490, 506). This, combined with mutagenesis/biochemical with gene expression profiles (462).
studies and improved computer modeling, has greatly en-
hanced our understanding of the relationship between Large-diameter sensory neurons (termed A␣ and ␤ affer-
structure and function of VGSCs. In simple terms, these ents) have peripherally projecting fibers that are thickly my-
channels can exist in three voltage-dependent conforma- elinated, conduct action potentials rapidly [⬎14 m/s in the
tional states. These are open, closed, and inactive, with each rat (207)], and in general respond to low-threshold mechan-
state determining channel conductance. At strongly hyper- ical stimuli. In the skin, A␤ fibers have specialized endings,
polarized membrane potentials, the channels will be closed for instance terminating around hair follicles or Merkel
and nonconducting. On depolarization, the voltage sensors cells (516). These have a low threshold for activation and
move outwards in a rotating movement. This pulls the pore possess either rapidly adapting or slowly adapting firing
open for a short period (⬍1 ms). The channel then enters properties. In deeper tissues, such as muscle, A␣ afferent
into an inactivated state via fast inactivation. A tethered, proprioceptors innervate muscle spindles.
cytoplasmic inactivation “gate” is located between do-
mains III and IV and comprises an IFM motif, flanked by A␤ nociceptors have been detected across several species,
glycine and proline residues. During fast inactivation, the where conduction velocities have been accurately deter-
flanking residues act as molecular hinges, allowing the IFM mined. For example, in both the rat and mouse, ~50% of
motif to swing and occlude the intracellular mouth of the A-fiber nociceptors conduct in the A␤ range (128, 268). In
pore, in a mechanism akin to a hinged lid (67, 465, 479).
the rodent, most A␦ and A␤ nociceptors were mainly clas-
Fast inactivation occurs within a millisecond time scale;
sified as high-threshold mechanoreceptive units, with only
however, a second distinct form of inactivation, known as
2% (in each group) also responding to noxious heat (128).
slow inactivation, acting over tens of seconds can occur in
response to prolonged depolarizations (470). The mecha-
A␦ afferents have intermediate cell size and axon diameter,
nisms involved in slow inactivation are not well under-
intermediate conduction velocity [2.2– 8 m/s in the rat
stood; however, crystallographic snapshots of the bacterial
(207)], and their axons are thinly myelinated. Many of these
Nav from Arcobacter butzleri may have provided some in-
sensory neurons are mechanical nociceptors with a high
sight into the mechanism. In a slowly inactivated state, the
mechanical threshold, although this group also includes
bacterial Nav undergoes asymmetrical arrangement of the
thermoreceptors that can respond to cooling and some hair
four Nav subunits. These morph into two pairs of confor-
mations, dramatically altering the structure of the central follicle afferents (D-hair afferents) (21, 268).
pore that is expected to render the channel nonconductive
(362). Ultimately, with hyperpolarization, the channel will C-fiber afferents have small-diameter cell bodies and thin
recover from inactivation (reprimes), return to the closed unmyelinated axons, resulting in a slow conduction velocity
state, and thus be available for activation. Four ␤-subunits [typically ⬍1.4 m/s in the rat (207)]. The majority of these
have been described. These have an NH2-terminal immu- afferents belong to nociceptive neurons. The term poly-
noglobulin-like domain, a single transmembrane segment, modal nociceptor was coined by Bessou and Perl (29) when
and a short intracellular segment (241). These form com- recording from cutaneous C-fiber nociceptors. The major-
plexes with ␣-subunits, ␤1 and 3 associating noncovalently ity of these respond to a broad range of noxious stimuli,
with ␣-subunits, and ␤2 and 4 form disulfide bonds with including high-intensity mechanical and thermal stimuli, as
␣-subunits. These ␤-subunits modulate the activation and well as chemical algogens [often termed CMH (mechano-
inactivation kinetics of VGSCs, regulate channel density at heat responsive C-fibers) in short hand]. Smaller subpopu-
the plasma membrane, and may also have a role in regulat- lations have more restricted sensitivity including CH, CM,
ing subcellular localization. and C mechano-cooling (CMC) units (302). Not all C fibers
are nociceptors, however, and in hairy skin in particular
there is group of low-threshold mechano-C fibers that have
B. Molecular and Functional Heterogeneity been characterized (284, 297). A␦- and C-fiber nociceptors
of Sensory Neurons have free nerve endings in their target tissues.
The cell bodies of sensory neurons are located in the dorsal These different functional classes of sensory neurons also
root ganglia (DRG) or trigeminal ganglia (TRG). They are have distinct developmental origins (305). The neurogen-
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BENNETT ET AL.
esis of low-threshold mechanoreceptors from neural crest neity than previously imagined, for instance Usoskin et al.
progenitors occurs before the neurogenesis of nociceptors (462) and Chiu et al. (77) have suggested 11 distinct groups.
(305). The survival and maturation/specification of differ-
ent functional classes of sensory neurons is developmentally Once a stimulus has been applied to a sensory terminal, a
regulated by neurotrophic factors (145). For example, pro- transduction element is activated, resulting in an ion flux.
prioceptive afferents are dependent on neurotrophin-3 VGSCs are then required to amplify this signal (termed a
(144, 156) and nociceptors on nerve growth factor (NGF) generator potential), which, once threshold is reached, trig-
(97) for survival during the embryonic period. In the mouse, gers a regenerative action potential, transmitting this infor-
two distinct groups of C-fiber nociceptors emerge in the mation to the spinal cord. In adulthood, the VGSCs Nav1.1,
postnatal period. There are those that express the NGF Nav1.6, Nav1.7, Nav1.8, and Nav1.9 (9, 40, 122, 175) are
receptor, TrkA and calcitonin gene-related peptide all expressed in primary sensory neurons (FIGURE 2). There
(CGRP), which are referred to as peptidergic nociceptors. is maturation to this expression pattern as the VGSCs
The second population are known as the nonpeptidergic Nav1.2, Nav1.3, and Nav1.5 (49, 160, 381) are expressed
nociceptors, and they lose their NGF sensitivity, instead during the embryonic period and subsequently downregu-
becoming responsive to glial derived neurotrophic factor lated. However, Nav1.3 can be reexpressed in adulthood
(GDNF) (26, 322). In addition, they upregulate the expres- following peripheral nerve injury (FIGURE 3) (discussed in
sion of P2X3 and are commonly detected by binding of the detail below) (475).
lectin IB4. Recent evidence suggests that there may not be
such a clear dichotomy between nociceptor subpopulations VGSC ␣-subunits have differing kinetics and distinct pat-
in human DRG neurons. In situ hybridization experiments terns of expression (both of which will be discussed in detail
found that nearly one-half of all TrkA-positive human neu- below), reflecting the functional groupings of sensory neu-
rons (176, 399). Large-diameter DRG neurons principally
rons coexpressed Ret, the receptor for GDNF, compared
express VGSCs, which are tetrodotoxin-sensitive (TTX-S)
with 23% in the mouse (391). In adulthood, sensory neuron
Nav1.1, Nav1.6, and Nav1.7 (40, 176, 217). A subset of
survival in a naive state no longer requires neurotrophic
these neurons also express the tetrodotoxin-resistant
signaling. However, these growth factors can have a major
(TTX-R) Nav1.8 (428), which could correspond to A␤ no-
influence on function, for instance through altering the ex-
ciceptors, which have been reported in the rat (128). In
pression of VGSCs (discussed further below).
comparison, small-diameter nociceptors express high levels
of the TTX-R VGSCs Nav1.8 and Nav1.9, as well as Nav1.7
The last 2 decades have seen significant advance in our
and Nav1.6 (45). The proposed role of the various VGSCs
understanding of the molecular machinery through which
in nociceptors is illustrated in FIGURE 3. The variations in
sensory neurons respond to external stimuli (132). Ligand-
VGSC expression are reflected by differences in the mor-
gated ion channels and G protein-coupled receptors trans-
phology of action potential waveform. In small-diameter
duce such stimuli. These are expressed in complex overlap-
nociceptors, the action potential is broader, with an inflec-
ping patterns by sensory neurons, and this expression tion on the falling phase as a consequence of greater TTX-R
determines the physiological heterogeneity of sensory neu- sodium current (largely mediated by Nav1.8) (FIGURE 3) (8,
rons. One example is the transient receptor potential (TRP) 9, 270, 394). The presence of an aromatic residue at the
family of ion channels. TRPV1 was the first to be linked to TTX-susceptibility-determining site in the extracellular
sensory transduction (66) and is a nonselective cation chan- linker between S5 and S6 in domain 1 (1-SS2) pore module
nel expressed by nociceptors activated by noxious heat, confers TTX sensitivity. The negative electrostatic potential
capsaicin (the active ingredient in chili peppers), and low of the aromatic resides (tyrosine and phenylalanine) attracts
pH. TRP channels are expressed throughout the entire neu- TTX (266, 410). This process is known as the cation-␲
ron, from peripheral and central terminals to the cell body interaction and increases the affinity of the toxin to its bind-
(64, 83). Sensory neurons express several TRP channels ing pocket within the outer vestibule of the TTX-S channels
(251). Each is attuned to detect specific stimuli, ranging (409). TTX-R channels, on the other hand, have either a
from innocuous warming (TRPV3) (324) to high tempera- serine or cysteine at this position, reducing the affinity of
tures (TRPV2) (65) and innocuous cooling (TRPM8) (310, TTX interacting with the channel by several orders of mag-
355) to noxious cold (TRPA1) (442). TRPA1 also responds nitude (433, 515). Site directed mutagenesis demonstrated
to various environmental irritants: e.g., cinnamon, mus- that replacing the hydrophilic serine to aromatic phenylal-
tard, acrolein (the active component of tear gas), and anine in the normally TTX-R Nav1.8 gave rise to a channel
wasabi. Single-cell RNA sequencing has enabled unbiased now highly sensitive to TTX (433), confirming the impor-
clustering methodologies to subdivide DRG neuronal pop- tance of interactions between TTX and aromatic residues in
ulations (77, 462). This has revealed that, at a molecular the pore-forming region of the ␣-subunit.
level, the subdivision of sensory neurons broadly agrees
with the classification based on cell size and correlates with One word of caution regarding extrapolating results from
neurotrophin receptor expression, but with more heteroge- rodent to human is that, while some features may be similar

FIGURE 2. The innervation pattern of mouse dorsal root ganglia (DRG) sensory neurons. DRG primary
afferent neurons are pseudo-unipolar that innervate peripheral targets, such as the skin and synapse centrally
in the dorsal horn of the spinal cord. Each subpopulation of DRG neurons has distinct central termination
patterns. Generally, unmyelinated neurons terminate in more superficial laminae compared with the myelin-
ated A␤ and A␦ fibers. All neuronal subpopulations express Nav1.1, Nav1.6, and Nav1.7 to some extent.
Small-diameter nociceptive neurons with unmyelinated C fibers can be broadly subdivided based on the
expression of calcitonin gene-related peptide (peptidergic) or the binding of IB4 (nonpeptidergic). Expression of
Nav1.9 is largely limited to the nonpeptidergic population (118). *A small population of large-diameter neurons
express Nav1.8 at a high level (217). Nav1.6 is clustered at the node of Ranvier in myelinated fibers where it
functions in saltatory conduction (62). There is also evidence that it contributes to continuous conduction in
nonmyelinated axons (46).
across species, others may differ considerably. For instance, come sensitized with a marked alteration in their stimulus-
there is a much larger overlap of NGF- and GDNF-depen- response function, demonstrating spontaneous activity,
dent nociceptors in humans, suggesting that the distinctive lowered thresholds to thermal and mechanical stimuli, in-
C-fiber subpopulations observed in rodents may not neces- creased response to suprathreshold stimuli, and after-dis-
sarily translate to humans (391). Correspondingly, expres- charges following removal of the stimulus (37, 204, 205,
sion of IB4, routinely used to identify the nonpeptidergic 267). Furthermore, there is a group of nociceptors present
C-fiber population in rodents, is questionable in human within multiple tissues that are ordinarily unresponsive (so-
DRG, with some studies demonstrating positive immuno- called silent nociceptors). However, following tissue in-
histochemical signal (351, 427), and others finding it is flammation, they develop a novel mechanosensitivity (311,
absent in cultured human DRG neurons (105). It is, there- 417). This sensitization is likely to be secondary to the re-
fore, important to bear in mind that the classification of lease of chemical mediators, cytokines, and growth factors
rodent DRG subpopulations and the molecular markers of from inflamed tissue, which can bind to receptors on noci-
these may not necessarily translate to the human. Further ceptors. Altered phosphorylation, trafficking, and expres-
anatomical and physiological classification of human DRG sion of transduction elements and ion channels, therefore,
neurons is needed to provide clarity on their subpopulations provide mechanisms for such sensitization.
and relevant markers.
Neuropathic pain arises as a consequence of a lesion or
C. Sensory Neurons and the Development of disease of the somatosensory nervous system. Neuropathic
Persistent Pain pain is associated with marked functional changes in pri-
mary afferents, as well as sensitization within the CNS.
We have described the response of nociceptors to an acute These gross changes in both the peripheral nervous system
noxious stimulus relevant to acute pain. However, in some (PNS) and CNS can lead to both spontaneous (which is
instances, the functional properties of sensory neurons can independent of a peripheral stimulus) and evoked pain.
change so radically after tissue injury that chronic pain can There may be such extensive changes in the stimulus-re-
develop. Chronic pain represents a major burden to human sponse function that a low-intensity stimulus, such as
health and has a number of defining features. In humans, brushing or cooling the skin, can now evoke pain (termed
this principally relates to duration, with pain lasting for brush and cold evoked allodynia, respectively) (245). After
more than 3 mo regarded as chronic. Another common nerve injury, ectopic activity develops in nociceptors, as
feature is that chronic pain is excessive to the degree of well as A␤-myelinated, nonnociceptive afferents (56, 292,
injury and, in many cases, is unfortunately resistant to treat- 472, 484). This ectopic activity is critically important in
ment (457). Following tissue inflammation (such as burn maintaining neuropathic pain, as recently shown by assess-
injury to the skin or joint inflammation), nociceptors being the efficacy of targeted local anesthetic nerve blocks in

BENNETT ET AL.
FIGURE 3. Contribution of voltage-gated sodium channels (VGSCs) to action potential generation. Represen-
tative action potential waveform recorded from a small-diameter nociceptive dorsal root ganglia neuron,
excited by a depolarizing current step of 200 pA is shown. The contribution of each VGSC to the generation of
an action potential in a nociceptive neuron is included. Inset: representative examples of fast TTX-S (sensitive)
currents from Nav1.3, Nav1.6, or Nav1.7; slow TTX-R (resistant) currents from Nav1.8, and persistent TTX-R
currents from Nav1.9.
reversing peripheral neuropathic pain (206). Ectopic activ- regards to peripheral sensory neurons, as in the CNS,
ity arises both within injured axons, neighboring uninjured Nav1.3 expression peaks during development (at embry-
axons, and at the level of the cell body (292, 472, 484). onic day 17) and then declines (23) to such an extent that
Peripheral axotomy of sensory neurons radically alters expression of Nav1.3 is undetectable in adulthood (475).
transcription with almost one-third of the genome demon-
strating altered expression (89, 357). Such changes include Despite the lack of expression in adult rat naive DRG neu-
the VGSCs, for instance the embryonic VGSC ␣-subunit rons, Nav1.3 is highly reexpressed in injured sensory neu-
Nav1.3 is reexpressed (475). Furthermore, VGSCs become rons. This reexpression occurs in both small- and large-
mislocalized following nerve injury (36, 211, 212, 359) as a diameter DRG neurons at mRNA (116, 475) and protein
consequence of altered axonal trafficking and changes in the level following sciatic nerve axotomy (38, 258). In contrast
nodal architecture. We will discuss these changes and the an- to the upregulation following peripheral axotomy, injury of
algesic potential of agents targeting individual VGSCs in the the central projection of sensory neurons via a dorsal root
subsequent sections. transection was not followed by any upregulation of
Nav1.3 immunostaining, this is, presumably as the periph-
eral source of neurotrophic molecules remains intact (38).
II. PAIN AND NaV1.3
Previous studies have demonstrated an accumulation of so-
dium channels (using pan-sodium channel antibodies) at
A. Functional Properties After Nerve Injury the neuroma of a severed nerve end (114, 140, 141). In
agreement with these earlier studies, while Nav1.3 was un-
Nav1.3, originally termed sodium channel III, was cloned detected along the proximal nerve trunk, Black et al. (38)
and sequenced in the 1980s from rat brain tissue (335). found a large accumulation of Nav1.3 immunoreactivity
Expression was reported primarily in the brain (445), and within axons just proximal to the ligature of a ligated and
early studies found that Nav1.3 mRNA levels greatly de- transected sciatic nerve. The accumulation of Nav1.3
clined in most regions of the brain during early postnatal within the proximal end of the transected fiber is consistent
development, with peak expression approximately at birth with studies demonstrating that axons within neuromas are
(24, 160). The only location where Nav1.3 expression re- hyperexcitable and can act as ectopic impulse generators
mains high in the adult is in sympathetic neurons (400). In (59, 412, 472).

The expression of Nav1.3 in the human is not well studied. To study the regulation of Nav1.3 expression, the effect of
A recent article found no difference in Nav1.3 transcrip- neurotrophic factors on injury-mediated dysregulation has
tional expression in human DRG (from male and female been investigated. GDNF is known to be neuroprotective,
donors ranging from 27 to 76 yr old) compared with adult preventing some axotomy-induced changes in injured neu-
mouse DRG (71). There were also no sex or age differences rons (26). GDNF treatment abolished the development of
in Nav1.3 expression. We could find no data on Nav1.3 mechanical and thermal hyperalgesia following partial
expression in human embryos. Therefore while we cannot nerve ligation (56). Significantly fewer spontaneous dis-
determine whether, in human DRGs, Nav1.3 undergoes a charges were observed in myelinated fibers within the dor-
perinatal downregulation as in the mouse, we can be sure sal root following GDNF treatment. On a biochemical
that the contribution of Nav1.3 to neuronal excitability in level, GDNF treatment prevented the upregulation of
the naive, uninjured state would be very minimal or nonex- Nav1.3, while also partially suppressing the downregula-
istent. Similarly, reexpression after injury in human DRG tion of Nav1.8 and Nav1.9 (56). In a study assessing the
neurons has not been determined. repriming kinetics of DRG neurons after sciatic nerve tran-
section, the characteristic reduction in the recovery time
Activation of Nav1.3 produces a fast-inactivating, rapidly- constant (due to the shift toward a more rapidly repriming
repriming current that can be blocked by TTX. The slow, current after injury) was observed in axotomized neurons;
closed-state inactivation can produce a substantial ramp however, this was partially normalized by intrathecal
current in response to small, slow depolarizations (98). The GDNF administration (279). Interestingly, NGF treatment
upregulation of Nav1.3 expression in the DRG following also partially restored the repriming kinetics to the same
nerve injury is rapid (detected ⬍24 h after injury) and is degree as GDNF (279). The coapplication of GDNF and
maintained for days to weeks, mirroring the development of NGF entirely reversed the effects of peripheral axotomy on
ectopic discharge (294, 295) and mechanical allodynia repriming kinetics (279). Furthermore, while single appli-
(218, 292). The increase in Nav1.3 expression is also par- cation of NGF or GDNF reduced the levels of Nav1.3
alleled by a dramatic switch in the electrophysiological mRNA, only combined application reduced the transcrip-
characteristics of the injured neurons. Axotomy decreased tional level of Nav1.3 to that comparable with the control
(279). The receptors for NGF and GDNF are expressed by
the number of small DRG neurons expressing a slow cur-
largely non-overlapping DRG subpopulations (18, 26, 254,
rent component (neurons were categorized as displaying a
322). Therefore, the additional reduction of Nav1.3 tran-
fast or slow current if the fast or slow component consti-
script levels by the combined NGF and GDNF treatment,
tuted ⬎70% of the total current) and increased the number
and the reversal of the rapidly repriming current, could be
with predominantly a fast current (104). The emergence of
due to the neuroprotective effect on a larger population of
a rapidly repriming TTX-S current following axotomy that
neurons.
is approximately fourfold quicker than in control neurons
would enable the C-fiber neuron to sustain repetitive firing,
contributing to an enhanced excitability. There is also a B. Association With Contactin and
good correlation between backfilled, axotomized small Trafficking of Nav1.3
neurons with a rapidly repriming TTX-S current (86%) and
those with Nav1.3 immunoreactivity (79%), again suggest- Contactin/F3 is a cell adhesion molecule that binds to the
ing that Nav1.3 is responsible for the change in electrophys- membrane via a glycosyl-phosphatidylinositol anchor.
iological properties (38). The axotomy-induced change in Contactin has also been shown to form a complex with
repriming kinetics following the reemergence of Nav1.3 full-length Nav1.3 by binding to both the NH2- and
could, therefore, provide a molecular mechanism for the COOH-terminal polypeptides (422). Coexpression of
hyperexcitability in injured neurons. Nav1.3 and contactin in human embryonic kidney (HEK)-
293 cells enhances membrane Nav1.3 expression and re-
While some groups have found that Nav1.3 is upregulated sults in a threefold higher current density, compared with
in small-diameter DRG neurons, others have demonstrated Nav1.3 transfection only (422). After peripheral nerve tran-
that the upregulation occurs preferentially in medium- and section, contactin levels were increased predominantly in
large-diameter neurons after axotomy (258). This is consis- DRG neurons with small-diameter cell bodies, which was
tent with the finding that spontaneous discharges develop paralleled by an increased immunoreactivity for contactin
mostly in A␦ and A␤ fibers after spinal nerve ligation (252, and Nav1.3 within axon terminals in the neuroma (422).
292, 313). There is, however, some evidence to suggest that The coexpression of contactin results in an increase in cur-
the upregulation of Nav1.3 occurs predominantly in nonin- rent density, likely to be caused by a greater insertion of
jured neurons. Lindia et al. (288) examined the coexpres- functional Nav1.3 channels in the cell membrane. Recruit-
sion of activating transcription factor-3 (ATF-3; a marker ment of contactin to the axonal surface has been shown to
of axotomized neurons) and Nav1.3 and found that only be activity dependent in hypothalamic neurons (364).
18% of Nav1.3-positive neurons also expressed ATF-3 af- Therefore, the hyperactivity in ligated sensory neurons may
ter SNI (spared nerve injury). drive the increased expression of contactin to the injury site.

BENNETT ET AL.
This could subsequently result in the heightened expression the conclusion that, despite the dysregulated expression of
of Nav1.3, both in the cell soma and at the cut axonal tip in Nav1.3, it is not required for the development of neuro-
axotomized sensory neurons. As discussed below, the reex- pathic pain behavior following peripheral nerve injury in
pression of Nav1.3 may have profound implications for the the mouse (331).
development of neuropathic pain and allodynia.
The use of mutant mice can be confounded by developmen-
C. In Vivo Knockout or Knockdown of tal compensation. When antisense oligonucleotides were
used to knockdown Nav1.3 expression, despite inducing a
Nav1.3 in Sensory Neurons
50% reduction in Nav1.3 immunoreactivity, no reversal in
A number of approaches have been used to test the role of mechanical allodynia was observed after SNI, suggesting
Nav1.3 in the development of ectopic activity and pain- multiple VGSCs likely contribute to neuropathic pain after
related behavior following nerve injury, with results differ- injury (288). However, alternative gene-silencing methods
ing according to the technology used. Nassar and colleagues have also been validated. Use of the highly specific second-
(331) generated a global Nav1.3-null mouse and found generation gene-silencing shRNAmir technology was able
acute and inflammatory pain behavior was normal com- to effectively reduce Nav1.3 expression in HEK cells stably
pared with littermate controls. Furthermore, the number of expressing Nav1.3 (FIGURE 4A) (405). This led to a signifi-
spontaneously active fibers and the development of me- cant reduction of voltage-gated sodium currents in these
chanical allodynia were the same in Nav1.3 knockout mice cells (FIGURE 4B). To study the effects of knocking down
compared with controls (331). Mechanical allodynia also Nav1.3 after nerve injury, delivery methods were first vali-
developed normally in strains in which Nav1.3 was selec- dated. Direct injection of Adeno-associated virus (AAV)-
tively ablated in nociceptors (using a Nav1.8 Cre) or CNS GFP into the L4 DRG (FIGURE 4C) resulted in a transduction
and PNS neurons (using a NFH-Cre). These results led to rate of ~45% (FIGURE 4D). Therefore, Nav1.3 shRNA was
FIGURE 4. Knockdown of Nav1.3 in rat dorsal root ganglia (DRG) attenuates spared nerve injury (SNI)-
induced neuropathic pain. A: Western blot analysis using pan-sodium channel antibody on HEK-Nav1.3 cell
lysates 48 h after treatment with a control scrambled sequence (shRNA-Sc) or shRNA directed against Nav1.3
(shRNA-C). ␤-Actin antibody was used to normalize the signal. B: voltage-gated sodium current was effectively
reduced 48 h after shRNA-C was introduced into HEK-Nav1.3 cells. C: schematic of experimental procedures,
showing SNI model where the common peroneal and tibial nerves are severed, leaving the sural nerve intact,
and the direct injection of AAV-GFP into the L4 DRG. D: L4 DRG injection of AAV-GFP resulted in a transduction
rate of 45 ⫾ 2%, determined by counting neuronal profiles (NeuN-positive) that also expressed GFP. In the
merged image, GFP transduced neurons are yellow. E: Von Frey measurements of mechanical thresholds from
hindpaws of SNI rats injected with shRNA-Sc or shRNA-C. A significant reversal of mechanical allodynia is
observed at days 20 and 25, compared with those injected with shRNA-Sc (*P ⬍ 0.05). Injections consisted
of 2 ⫻ 1 ␮l in two different sites for each DRG, at a flow rate of 0.4 ␮l/min, using a custom-designed sharp
glass micropipette (20- to 30-␮m tip diameter) mounted on a 10-␮l Hamilton needle. [Adapted with permission
from Samad et al. (405).]

incorporated into an AAV vector and directly injected into ity and the development of chronic, neuropathic pain.
the L4 DRG on the same day that SNI surgery was per- These findings may seem to contrast the finding that me-
formed (FIGURE 4C). While direct injection into the DRG chanical allodynia following nerve injury was unchanged in
has been shown to cause transient mechanical hypersensi- mutant mice in which Nav1.3 is ablated (either globally or
tivity, this had no additional impact on mechanical within an NFH Cre-dependent manner, which would include
drawal thresholds associated with SNI. The onset of me- CNS neurons; see discussion above) (331). There are, how-
chanical allodynia developed normally in SNI rats injected ever, differences in the species (rat vs. mouse) and neuro-
with Nav1.3 shRNA; however, there was a significant re- pathic pain model (CCI vs. SNI) used in these reports,
covery of mechanical hypersensitivity compared with SNI which may account for these variances.
rats injected with scrambled shRNA after 10 –20 days (FIG-
URE 4E) (405). This study, therefore, provides important Patients with spinal cord injury (SCI) report moderate to
evidence for a role of Nav1.3 in the development of neuro- severe pain, and experimental contusion in rodents can pro-
pathic pain. duce long-lasting neuropathic pain. After SCI, analysis of
dorsal horn neurons reveals a hyperexcitable phenotype
The same group went on to validate the use of shRNA that is associated with the upregulation of Nav1.3 expres-
knockdown of Nav1.3 in a model of painful diabetic neu- sion in dorsal horn neurons similar to that seen in the pe-
ropathy. Streptozotocin (STZ)-induced diabetic models of ripheral DRG after nerve injury. Selective knockdown of
neuropathic pain also result in upregulation of Nav1.3 in Nav1.3 decreased expression of Nav1.3 mRNA and protein,
sensory neurons similar to that seen after traumatic nerve reduced hyperexcitability of dorsal horn neurons, and re-
injury (93). Using the same shRNA construct as validated in duced mechanical allodynia and thermal hyperalgesia after
their earlier paper (405), but now administered intrathe- SCI (193). This study was the first to demonstrate Nav1.3
cally, the same group demonstrated a significant knock- reexpression in second-order neurons can trigger a func-
down of Nav1.3 mRNA (446). At 3 wk postinjection, the tional shift in central excitability that can be linked to the
shRNA-Nav1.3-treated animals showed a significant rever- onset of neuropathic pain.
sal of nerve injury-induced mechanical allodynia (446).
SCI can also trigger supraspinal changes in VGSC expres-
sion in thalamic neurons (195). Twenty-eight days after
D. Expression and Function of Nav1.3 in SCI, Nav1.3 immunostaining signal was greatly increased in
Second- and Third-Order Neurons neurons within the ventral posterolateral nucleus of the
thalamus. Recordings from ventral posterolateral neurons
Peripheral nerve injury can also result in changes in Nav1.3 from SCI rats demonstrated a high rate of spontaneous
expression within dorsal horn neurons. Ten days after the activity that was independent of ascending afferent input
chronic constriction injury (CCI; in which chromic gut su- (195). Antisense targeting of Nav1.3 significantly reduced
tures are loosely tied around the sciatic nerve) model of Nav1.3 expression within the thalamus and reversed the
nerve injury performed in the rat, lumbar spinal cord sec- increase in spontaneous activity within these neurons. The
tions were probed for Nav1.3 mRNA, and a strong hybrid- increased barrage from peripheral DRG neurons after SCI
ization signal was clearly present in the ipsilateral side may trigger molecular changes in supraspinal locations;
within neurons in dorsal horn laminae I-II (194). Further- however, the enhanced spontaneous activity continued
more, neurons expressing Nav1.3 were localized more in even after complete spinal cord transection. This indicates
the medial region of the dorsal horn, the known projection constant ascending input is not essential to maintain hyper-
site of primary afferent neurons innervating the hindlimbs. excitability in the thalamus, suggesting this region may be
Upregulation of Nav1.3 mRNA coincided with an increase able to act as an independent intrinsic pain generator.
in Nav1.3 immunostaining in the dorsal horn that was co-
localized with staining for the neurokinin-1 receptor (the
receptor for substance P), a marker for nociceptive dorsal E. Nav1.3 as a Therapeutic Target
horn projection neurons. Oligodeoxynucleotide (ODN)
knockdown of Nav1.3 specifically in the dorsal horn The upregulation of Nav1.3 in DRG neurons and indeed
(ODNs were not detected in DRG neurons) prevented the second- and third-order neurons in central pain pathways
upregulation of Nav1.3 mRNA in the spinal cord and re- following nerve injury, the correlation of this reexpression
sulted in a loss of Nav1.3 immunoreactivity in neurokinin-1 with neuronal hyperexcitability, and the fact that this chan-
receptor-positive neurons. Knockdown of Nav1.3 mRNA nel could support higher firing frequency due to rapid rep-
in the dorsal horn resulted in a behavioral reduction in riming are suggestive of a role of Nav1.3 in the pathogenesis
mechanical allodynia and thermal hyperalgesia, both of of peripheral neuropathic pain. This hypothesis has been
which were restored upon cessation of ODN administra- tested in a number of different ways, including ODN or
tion (194). Therefore, expression of Nav1.3 is upregulated shRNA-mediated knockdown and mutant mice lacking
in second-order dorsal horn neurons after peripheral injury, Nav1.3 with, at times, conflicting results. Greater efficacy
which could be a link between the peripheral hypersensitiv- has been seen using the knockdown-mediated approach

BENNETT ET AL.
than in using mutant mice, which could relate to differences Nav1.7 is involved with vasopressin release in response to
in pain model, species, and/or developmental effects. osmotic stress. Nav1.7 is also expressed in the hypotha-
Nav1.3 is a potential target for analgesics. The homology lamic arcuate nucleus, dorsal medial hypothalamic nu-
with other sodium channels, including 85% homology with cleus, and paraventricular hypothalamic nucleus in
Nav1.2, however, limits the likelihood of developing a which it has a role in synaptic input integration of AGRP
Nav1.3-specific blocker (452). Furthermore, it would be and POMC neurons and energy homeostasis (57). Al-
important to establish if Nav1.3 is required for the long- though Nav1.7 mRNA was prominently detected in the
term maintenance of neuropathic pain. paraventricular and supraoptic hypothalamic nuclei in
rodents, the signal is considerably weaker in monkey and
human brains, questioning the role of Nav1.7 in hypo-
III. PAIN AND NaV1.7 thalamic control of autonomic function (5).
A. Preclinical Investigations 2. Expression within the PNS: vagal sensory neurons
Early serological studies suggested different molecular Nav1.7 is also abundantly expressed in vagal sensory neu-
properties between sodium channels extracted from central rons. The cell bodies of these neurons are located in the
and peripheral regions (482). These molecular differences nodose ganglion, from which vagal afferents project
helped to identify Nav1.7 as the major sodium channel ex- throughout the body to innervate visceral tissues. A large
pressed in peripheral neurons (455). Northern blot expres- proportion of these neurons project capsaicin-sensitive no-
sion analysis revealed positive signal in the superior cervical ciceptive C fibers that innervate the respiratory tract. Like
and DRG ganglia; however, no transcripts were detected in the somatosensory system, these capsaicin-sensitive neu-
the heart, skeletal muscle, and brain, confirming Nav1.7 is rons can also be activated by inflammatory mediators and
preferentially expressed in the PNS. noxious physical injury (273); however, this activation does
not cause pain. Instead, parasympathetic reflex pathways
1. Expression within the CNS are activated, resulting in bronchospasm and mucus secre-
tion, changes in breathing patterns, and an urge to cough.
Two other nodose neuronal populations innervate the re-
Despite the original finding that Nav1.7 is predominantly
spiratory system, including non-nociceptive, capsaicin-in-
localized in the PNS, it has subsequently been detected in
sensitive, low-threshold mechanoreceptors that innervate
distinct regions of the CNS. Olfactory sensory neurons
the lungs; and touch-sensitive, cough-evoking fibers that
(OSNs) are one such location. These neurons exhibit TTX-S
project to the larynx, trachea, and extrapulmonary bronchi
sodium currents (459) and have unmyelinated axons, which
(273). TTX-S sodium channels play a crucial role in vagus
synapse on mitral cells in the olfactory bulb (165). Nav1.7 is
nerve conduction, with 1 ␮M TTX almost entirely blocking
the major VGSC found in both the mouse and rat olfactory
the compound action potential (158). Selective inhibition of
epithelium, with low levels of Nav1.3, Nav1.6, and Nax also
Nav1.7 synthesis led to ⬎60% reduction in the fast TTX-S
present (6). Analysis of the axonal branch of OSNs has
sodium current in dissociated nodose ganglion neurons,
shown Nav1.7 is expressed in punctate structures within
suggesting Nav1.7 plays a crucial role in determining neu-
olfactory bulb glomeruli. Furthermore, there was no colo-
ronal excitability in these neurons. Knockdown of Nav1.7
calization with the dendritic marker, MAP2, suggesting
in the nodose ganglion almost completely abolished citric
Nav1.7 is restricted to presynaptic OSN. The high expres-
acid-induced cough while having no effect on respiration
sion of Nav1.7 in OSNs compared with other sodium chan-
rate (325).
nels suggests it has a key functional role in these neurons
(477). This is consistent with the marked impairments in
olfaction in rodent and humans as a consequence of ho- 3. Expression within the PNS: DRG sensory neurons
mozygous loss-of-function mutations in this gene (see be-
low). Much of the current literature on Nav1.7 centers on the
high level of expression in peripheral sensory neurons.
Within the rat hypothalamic supraoptic nucleus, several Nav1.7 mRNA has been detected in DRG neurons of all
TTX-S sodium channels have been characterized, including sizes (40); however, antibodies raised against Nav1.7 show
Nav1.2 and Nav1.6. Recently, Nav1.7 has been demon- greater binding in small rather than large DRG neurons in
strated to be expressed in magnocellular neurosecretory adult rodents (129). Electrophysiological analysis com-
cells of the supraoptic nucleus, that colabeled with vaso- bined with immunocytochemistry demonstrated that
pressin and oxytocin (43). The level of Nav1.7 within these Nav1.7 staining intensity was negatively correlated with
hypothalamic neurons was also shown to be dynamic, sig- dorsal root conduction velocity and positively correlated
nificantly increasing by approximately twofold as a result of with action potential duration in the guinea pig, both sug-
salt-loading the rodent’s drinking water (43). Due to its gesting it is preferentially expressed in small nociceptive
expression within vasopressin neurons, it is possible that neurons (129). In support of these findings, an upregulation

of Nav1.7 immunoreactive clusters was detected in neuro- at prepulse potentials between ⫺130 and ⫺10 mV, then
mas from human patients with a range of nerve injuries. measuring the peak current amplitude elicited by 20-ms test
The study suggests a heightened increase of Nav1.7 immu- pulses to ⫺10 mV. The midpoint of the h-⬁ curve, where
noreactivity in painful compared with nonpainful neuro- 50% of channels are available, was significantly more neg-
mas (271), highlighting the role of Nav1.7 in nociception. ative for Nav1.7 (⫺78 mV) compared with Nav1.4 (⫺72
mV) (103). To characterize the properties of Nav1.7 in a
To study regional localization of Nav1.7 within rat DRG native system, Herzog and colleagues (214) expressed
sensory neurons, immunohistochemistry against Nav1.7 Nav1.6 and Nav1.7 in mice DRG neurons. Both channels
was combined with additional markers of neuronal sub- were mutated into a TTX-R derivative (substituting the
populations. Approximately 63% of peripherin-positive C aromatic tyrosine to serine at position 371 for Nav1.6, and
fibers (190) and 15% of neurofilament-200-positive, my- 362 for Nav1.7), and expressed in DRG neurons from
elinated A fibers co stained for Nav1.7 in the soma (FIGURE Nav1.8 knockout mice. This allowed identification of the
5B). In nociceptors, Nav1.7 expression was observed in current from transfected channels against the background
65% of the nonpeptidergic, IB4⫹ population and 58% of in which other endogenous TTX-S sodium currents can be
the CGRP-positive neurons (FIGURE 5C) (42). Within the blocked with TTX and in which the main endogenous
skin, the free nerve terminals of unmyelinated sensory neu- TTX-R current in cultured DRG neurons (provided by
rons displayed strong Nav1.7 immunoreactivity (FIGURE Nav1.8) is absent (214). An analogy by which VGSC func-
5A) (42). In the peripheral axon, Nav1.7 was detected in tion can be conceptualized is that it is regulated by two
~27% of unmyelinated, peripherin-positive fibers (FIGURE gates, the activation and inactivation gate. Conduction oc-
5D) and was particularly concentrated at the node of Ran- curs when both gates are open, and inactivates when a
vier of small-diameter myelinated fibers (FIGURE 5E). component of the inactivation gate (a cytoplasmically
located inactivation particle) diffuses in to occlude the inner
In the centrally projecting axon terminal, Nav1.7 immuno- channel pore after depolarization has activated the S4 heli-
reactivity was colocalized with 30% of peripherin-positive ces. The inactivation gate usually closes when the activation
fibers in the dorsal root. Within the superficial dorsal horn, gate is open, known as open-state inactivation; however, it
Nav1.7 was abundantly detected in lamina I, lamina IIo,
can also close before the channel is able to conduct, a pro-
and lamina IIi within primary afferent terminals (FIGURE
cess known as closed-state inactivation (17). Herzog et al.
5F). All NeuN-positive cells within the dorsal horn were
(214) found that the development of closed-state inactiva-
negative for Nav1.7 immunoreactivity, strongly suggesting
tion was far slower for Nav1.7 than Nav1.6 (~150 ms com-
that postsynaptic second-order neurons do not express this
pared with 20 ms, respectively, at ⫺70mV). Based on the
sodium channel. Instead, Nav1.7 was extracellular to these
slow kinetics of closed-state inactivation, Cummins and
NeuN-positive dorsal horn neurons and highly colocalized
colleagues (103) predicted that a proportion of Nav1.7
with synaptophysin, a marker of synapses, indicating that
channels would remain available for activation during a
Nav1.7 is only expressed in presynaptic terminals (42).
slow-ramp depolarization. This was tested by slowly depo-
larizing from ⫺100 to ⫺50 mV and then stepping to 0 mV
In conclusion, Nav1.7 is expressed to a greater extent in
nociceptive than nonnociceptive sensory neurons. The ex- to record how much current remained available for activa-
pression is located along the entire length of these neurons tion. After a short ramp (1 mV/ms, 50-ms total duration),
from epidermal free nerve endings to presynaptic central 76% of the current remained available, and, even when
terminals, suggesting an important functional role in noci- lengthening the ramp to 250 ms (0.2 mV/ms), 36% re-
ceptive processing. mained available from Nav1.7, which was significantly dif-
ferent from Nav1.4 (37% remained at 50 ms and 10%
4. Biophysical properties of Nav1.7 remained at 250 ms) (103). Similarly, when expressed in
mouse DRG neurons, Nav1.7 elicited significantly larger
A) CHANNEL ACTIVATION AND INACTIVATION. When expressed in subthreshold current than Nav1.6 in response to slow-ramp
HEK-293 cells, Nav1.7 displayed similar activation proper- depolarizations (214). Therefore, due to the slow onset of
ties to another TTX-S sodium channel, Nav1.4. For exam- closed-state inactivation, Nav1.7 is able to produce a robust
ple, the time course of activation was almost identical in ramp current, in response to subthreshold, slowly depolar-
transfected cells, with the midpoint of activation being izing stimuli. Amplification of slow depolarizations caused
⫺25.8 mV for Nav1.7 and ⫺27.0 mV for Nav1.4. Similarly, by natural stimuli, such as through activation of a ligand-
the time constants for deactivation (repolarization phase) gated ion channel, can increase the probability of the neu-
were similar between these sodium channel subtypes. How- ron reaching firing threshold. This suggests that Nav1.7
ever, when inactivation kinetics were assessed, Nav1.7 cur- may, therefore, amplify generator potentials and act as a
rents inactivated with a time constant of 0.77 ms at 0 mV, threshold channel with an important role in impulse initia-
whereas Nav1.4 inactivated significantly faster with a time tion. Consequently, the sodium currents through this chan-
constant of 0.51 ms (103). The voltage dependence of nel play an important role in impulse initiation and
steady-state inactivation was examined by holding the cells pacemaking.

BENNETT ET AL.

B) CHANNEL REPRIMING.The firing rate of a neuron is limited by 5. Splice variants of Nav1.7

the channel’s recovery rate from fast inactivation (reprim-
ing), which differs greatly in large vs. small DRG neurons. Nav1.7 is subject to alternative splicing at two distinct sites,
The time constant for TTX-S sodium channel repriming in which may have important implications for the biophysical
small neurons is ~150 ms at ⫺80 mV, compared with only properties of the channel. The gene encoding Nav1.7,
25 ms in large neurons (149). An explanation for this dif- SCN9A, was originally reported to have 26 exons, with the
ference could be that the kinetics of repriming differ in large splice sites located in exons 5 and 11. However, with the
vs. small DRG neurons, which is conveyed by distinct ex- discovery of an additional exon in the 5=-UTR, exon num-
pression patterns of sodium channels in these neurons. bers increased to 27. Therefore, we refer to splice sites in
Nav1.6 is highly expressed in large DRG neurons, while exons 6 and 12 to take this into account. The splice site in
only low levels can be detected in small neurons; conversely, exon 6 (FIGURE 6A) is located in the region that encodes the
Nav1.7 exhibits greater expression in small DRG neurons. S3-S4 extracellular linker and the S4 voltage sensor of the
Indeed, repriming kinetics were much faster for Nav1.6 first domain (181, 376). The second conserved splice site is
than Nav1.7 (214). Nav1.6 alone closely matched those of in exon 12, which differs by the presence (12L) or absence
TTX-S currents in large DRG neurons, whereas Nav1.7 (12S) of an 11-amino acid sequence in the intracellular
repriming kinetics were similar to those measured in small linker between domain I and II (FIGURE 6C) (72, 376). Both
neurons (214). Based on this evidence, the differential ex- splices sites are subject to developmental regulation; the
pression of Nav1.6 and Nav1.7 could underlie the distinct two mutually exclusive exon 6 alternates are named accord-
repriming kinetics of TTX-S sodium channels in large and ingly [6A (adult) or 6N (neonatal)]. Whereas 6N is more
small neurons, respectively. However, both Nav1.6 and prevalent neonatally, there is similar expression of the two
Nav1.7 have been found to be expressed, albeit at various isoforms during adulthood (FIGURE 6B). The long version of
levels, in neurons of all sizes (46, 407, 455), suggesting that exon 12 is more prevalent in adult compared with neonatal
these individual channels may not exclusively be responsi- neurons (FIGURE 6D) (78).
ble for the repriming kinetics in large and small DRG neu-
These splice variants of Nav1.7 have also been detected in
rons, respectively. The expression of auxiliary proteins,
human DRG neurons, although there are clear differences
which have close, functional interactions with sodium
in expression levels when comparing human to rat. For
channels, can also impact on the rate of repriming. One
example, 80% of rat SCN9A transcripts include exon 12L,
such example is a member of the fibroblast growth factor whereas this variant made up 45% of overall human DRG
homologous factors (FHF). FHF2A is expressed in the DRG SCN9A transcript levels. However, the significance of this
and can bind directly to the COOH-terminus of Nav1.6, species difference is unknown (376). The alternative iso-
strongly slowing repriming and leading to the accumulation forms are reported to impact on the biophysical properties
of inactive channels during repetitive stimulation (402). of Nav1.7. For example, 6A results in a significant slowing
FHF2A was colocalized with Nav1.6 in many small-diam- of inactivation at negative potentials, resulting in larger
eter neurons and could not be detected at the node of Ran- current amplitudes evoked with slow ramp depolarizations
vier in larger diameter, myelinated neurons, suggesting the (72). Protein kinase A and C (PKA, PKC) phosphorylation
association of Nav1.6 with FHF2A may influence the rep- sites are important in modulating the activity of sodium
riming kinetics of TTX-S channels in small neurons. channels, including Nav1.7 (469). Furthermore, increased
FIGURE 5. Nav1.7 expression extends from peripheral to central terminals. A: expression of Nav1.7 in glabrous skin. PGP9.5 (green)
intraepidermal nerve fibers (IENF) branch from nerve bundles (arrowheads) at dermis/epidermis boundary (dotted line) and ascend in the
epidermis (arrows). PGP9.5-positive IENF exhibit Nav1.7 (red) immunolabeling. Inset: both IENF extending to stratum granulosum (more
superficial) and to the stratum spinosum exhibit Nav1.7 labeling. B and C: expression of Nav1.7 in dorsal root ganglia (DRG) neurons. B: DRG
sections were reacted to antibodies against peripherin, neurofilament 200 (NF), and Nav1.7. Peripherin-positive (green) neurons are generally
of small diameter (⬍30 ␮m), and most exhibit colocalization (yellow) with Nav1.7 (red). A few small peripherin-negative neurons display robust
Nav1.7 immunolabeling (arrows). Neurofilament 200 (NF)-positive (blue) neurons are generally larger than peripherin-positive cells, and most do
not display colocalization with Nav1.7. A few smaller NF-positive cells exhibit Nav1.7 immunolabeling (magenta). C: DRG sections were reacted
with IB4-488 and antibodies to calcitonin gene-related peptide (CGRP) and Nav1.7. Virtually all IB4⫹ (green) neurons display colocalization
(yellow) with Nav1.7. Similarly, nearly all CGRP-positive (blue) neurons exhibit colocalization (magenta) with Nav1.7 (red). Few DRG neurons
(arrow) display colocalization of IB4, CGRP, and Nav1.7. D and E: expression of Nav1.7 in sciatic nerve. D: numerous peripherin-positive (green)
unmyelinated fibers are immunolabeled in sciatic nerve, and these fibers exhibit extensive colocalization (yellow) with Nav1.7 (red). Inset: at
increased magnification, peripherin-positive (green) fiber displays colocalization (yellow) with Nav1.7. E: nodal regions in sciatic nerve were
identified by paranodal caspr (green) labeling. Nav1.7 (red) immunolabeling at a node is displayed by a small-diameter (⬍1 ␮m) myelinated fiber.
Not all small-diameter myelinated fibers exhibit Nav1.7 labeling at nodes. F: Nav1.7 expression in spinal cord dorsal horn. Sections of spinal cord
were labeled for IB4 (green), CGRP (blue), and Nav1.7 (red). IB4 labeling is prominent in lamina IIi, whereas CGRP immunoreactivity is localized
to lamina I and IIo. Robust Nav1.7 immunolabeling is present within lamina I and II and exhibits colocalization with IB4 (yellow) and with CGRP
(magenta). There is limited overlap of IB4 and CGRP in the superficial lamina. [Adapted with permission from Black et al. (42).]

BENNETT ET AL.
FIGURE 6. Splice variants of SCN9A. A: schematic of exon 6 splice sites, showing the neonatal (E6N; orange)
and adult (E6A; green) isoforms. Exons 5 and 7 are shown in blue. The top and bottom images show the
transcript after intronic regions had been removed and the inclusion of either E6N (orange lines) or E6A (green
lines). B: quantification of exon 6 splice variant expression at neonatal and adult ages in rat. C: schematic of
exon 12 splice sites. Inclusion of an 11-amino acid sequence (burgundy) gives rise to the long splice variant
(E12L; bottom image), whereas exclusion of this sequence gives rise to the short splice variant (E12S; top
image). D: quantification of exon 12 splice variant expression at neonatal and adult ages in rat, demonstrating
E12L is more common during adulthood. B and D: real-time PCR was used to quantify the expression of the
splice variants. Raw data were normalized against the 18S rRNA housekeeping gene. [B and D are adapted
with permission from Choi et al. (78).]
kinase activity in nociceptive fibers (4, 125, 257) and dorsal of the ␤1-subunit with exon 12 variants did not affect chan-
horn neurons have been reported during hyperalgesia (126, nel activation; however, the voltage dependence of inacti-
238, 306). Significant changes in the relative proportions of vation was significantly shifted by the presence of this aux-
the alternatively spliced variants are observed after neuro- iliary subunit. In channels containing the 12S variant, the
pathic injury in rat DRG. Transcripts containing 12S in- shift in inactivation averaged ⫹4.5 mV, whereas the 12L
creased in relative abundance after SNL, indicating this iso- variant induced a significant shift averaging ⫹14.6 mV
form may be involved in neuropathic pain (376). Electro- (measured at the midpoint of inactivation). The shift in the
physiological recordings of tsA201 cells transfected with voltage dependence of inactivation introduced by the coex-
the human splice variants revealed no biophysical differ- pression of the ␤1-subunit with the 12L spice variant influ-
ences between 12S and 12L; however, the 12S variant could ences channel availability. At ⫺80 mV, ⬎60% of channels
be modulated by 8-bromo-cAMP. PKA-mediated phos- are inactive in the absence of the ␤1-subunit, shifting to only
phorylation after cAMP treatment resulted in a negative 20% when the ␤1-subunit is coexpressed. The effect ␤1 has
shift of the activation curve of variants containing exon on inactivation with variants containing 12L suggests an
12S, whereas those with 12L were unaffected (72). The interaction between the auxiliary subunit and the intracel-
increased expression of the 12S variant combined with en- lular loop between domains I and II (157).
hanced cAMP after injury could decrease action potential
threshold, thereby increasing neuronal hyperexcitability. 6. Evolution and species differences in Nav1.7
Sodium channel ␣-subunits are usually associated with one Acid is an algogenic stimulus that is commonly associated
or more auxiliary ␤-subunits that modulate expression and with pain and inflammation. Acid nociception is common
localization of the channel, as well as gating kinetics (67). across all vertebrates examined, with one exception. Re-
Using heterologous expression in HEK-293 cells, splice markably, it is entirely absent in the African naked mole rat
variants of exon 6 were preferentially shown to determine (435). The nociceptors of these mammals possess some ex-
the degree to which the ␤1-subunit altered the voltage de- ceptional characteristics. TRPV1 and acid-sensing ion
pendence of activation. Variants containing 6N produced a channels (ASICs) are well characterized acid sensors, both
five times larger hyperpolarizing shift in activation com- of which were found to be present in the naked mole rat and
pared with those with the 6A variant (157). Coexpression to have similar proton sensitivity compared with the mouse

orthologs. In an attempt to understand the mechanism for In addition to the loss of inflammatory pain, Nassar and
acid insensitivity, Smith and colleagues (436) focused on colleagues discovered that global knockout of Scn9a results
VGSCs, which are known to be inhibited by protons. Pro- in neonatal lethality due to a lack of feeding (332). To
tons reduce sodium conductance by lowering channel con- investigate this further, Nav1.7 was conditionally ablated in
ductance and neutralizing surface charges to shift the volt- cells expressing olfactory marker protein, which includes all
age dependence of gating in the depolarized direction (256). OSNs. They found that milk was absent from newborn
Application of a pH 6 solution to naked mole rat DRG pups, suggesting that lethality was due to failure to suckle as
neurons resulted in a 63% decrease in voltage-gated inward a consequence of anosmia. This led to investigations to
conductance, which was significantly greater than the 42% determine the role of Nav1.7 in olfaction and the finding
observed in mouse neurons (436). Furthermore, the po- that Nav1.7 is expressed by OSNs and is required presyn-
tency of pH 4 and pH 6 evoked inhibition of mechanically aptically for neurotransmitter release at the olfactory glom-
induced C-fiber activity in the naked mole rat was signifi- erulus (477).
cantly greater than in mouse neurons. The molecular basis
of the acid sensitivity lies with a specific variant of Nav1.7. Whereas global deletion of Nav1.7 has been described as
A highly conserved positively charged motif in the pore loop lethal due to the lack of feeding, a recent report has over-
region between S5 and S6 in domain IV was found to be come this by developing genetic and animal husbandry
negatively charged in the naked mole rat (436). This charge strategies to enable the survival of global Nav1.7 knockout
difference renders the naked mole rat Nav1.7 more sensitive animals to adulthood. Heterozygous Nav1.7 mice on a
to proton block. Therefore, despite the presence of acid C57BL/6 background were outcrossed to two different
sensors in the form of TRPV1 and ASICs, the depolarizing mouse strains reported to provide better maternal care, and
input through these channels is unable to overcome the hand fed two to three times a day. These methods resulted
block on Nav1.7 by H⫹, meaning acid evoked action poten- in consistently obtaining global Nav1.7 knockout adult
tials are not evoked. This phenotype is of major biological mice (183). Behavioral testing of these mice showed gross
importance for the naked mole rat; they live in subterranean reductions in nociceptive sensitivities. All knockout mice
burrows that experience unusually high CO2 levels able to lasted to the predetermined maximum latency time on the
drive tissue acidosis. Similarly, Nav1.7 in the cave-roosting hot plate without displaying any pain behaviors. Knockout
microbat, which also experiences high CO2 levels, was mice also displayed no mechanical hypersensitivity to a tail-
found to also have a negatively charged motif in the same clip stimulus, despite normal responses to nonnoxious stim-
pore loop region (436). Therefore, convergent evolution uli with von Frey hairs. Responses to intraplantar formalin
may have selected Nav1.7 as a target to modulate various were also dramatically reduced, with almost no detectable
stimulus transduction routes, with acid nociception being responses in phase II (183). These data highlight the impor-
an excellent example in the African naked mole rat. tance of Nav1.7 for the transmission of noxious stimuli to
second-order neurons. The overall density of the TTX-S
7. Examination of Nav1.7 function in pain signaling sodium current was lower in knockout mice, demonstrating
using rodent models that not all TTX-S current is encoded by Nav1.7 in mouse
DRG neurons. The frequency of mechanically evoked ac-
To assess the physiological role of Nav1.7, the Cre-recom- tion potentials recorded from single fibers in a saphenous
binase-loxP system was used to specifically ablate exons 14 nerve, skin nerve preparation were also reduced but not
and 15 of Scn9a, which encodes most of domain II of mouse absent in these animals (183). This would imply that
Nav1.7. Ablation was achieved preferentially in nociceptive Nav1.7 contributes to, but is not exclusively responsible for,
neurons by crossing a Nav1.8 Cre mouse with Nav1.7fl;fl axonal transmission, and that insensitivity to noxious stim-
mice (332). Withdrawal latencies to noxious thermal stimuli is not simply due to an inability to transduce such stim-
uli applied by the hot plate were unaffected in the condi- uli.
tional knockout mice. However, these mice were ~20% less
sensitive to radiant heat (using Hargreaves apparatus). Sen- To determine the role of Nav1.7 in modality-specific sen-
sitivity to noxious mechanical stimulation was significantly sory responses, Minett and colleagues (317) specifically ab-
reduced by ~50% when tested with a Randall-Selitto appa- lated Nav1.7 in defined neuronal subpopulations. A num-
ratus; however, no differences were observed to von Frey ber of Cre expressing mouse lines were used to selectively
hair stimulation. In response to inflammatory pain, condi- target populations of neurons. For pan-DRG gene recom-
tional knockout of Nav1.7 in nociceptors resulted in re- bination, a mouse in which Cre recombinase is expressed
duced nocifensive responses. Strikingly, Nav1.7 mutant under the Advillin promotor was bred with Nav1.7fl;fl mice
mice were almost entirely unaffected by injection of com- (Nav1.7Advill). For nociceptor recombination, a Nav1.8-Cre
plete Freunds adjuvant (CFA) when assessed for thermal mouse was used [Nav1.7Nav1.8, as previously described by
hyperalgesia and mechanical allodynia (332), which point Nassar et al. (332), see above]. Finally, for both sensory and
toward a major role of Nav1.7 in the mediation of inflam- autonomic neuron recombination, a Wnt1-Cre mouse line
matory pain. was used (Nav1.7Wnt1) (317). In all three mice lines, nox-

BENNETT ET AL.
ious mechanosensation tested by the Randall-Selitto test expression in Nav1.8-negative DRG neurons appears to be
was markedly reduced to a significant extent compared crucial for mechanical allodynia (316).
with littermate controls. These results suggest that deleting
Nav1.7 within Nav1.8 neurons is sufficient to abolish me- After spinal nerve transection (SNT; in which a lumbar
chanical pain behavioral responses. In response to noxious spinal nerve contributing to the sciatic nerve is transected),
heating by Hargreaves apparatus (a test of spinal with- the development of the prominent cold and mechanical hy-
drawal reflex) and cooling by acetone, Nav1.7Advill and persensitivity is thought to be related to the invasion of the
Nav1.7Wnt1 mice were both significantly hyposensitive, yet DRG by postganglionic sympathetic axons, resulting in
responses from Nav1.7Nav1.8 mice remained unchanged sympathetically mediated pain (372). Although the contri-
compared with littermate controls in response to both stim- bution of the sympathetic nervous system to hyperalgesia
uli. The unchanged responses of Nav1.7Nav1.8 mice using after SNT has been questioned, as one study found, sympa-
Hargreaves apparatus is at odds with earlier findings using thectomy after spinal nerve lesion did not reverse neuro-
the same mice, which found ~20% less sensitivity than in pathic behavior (384). Nevertheless, after SNT, sympa-
control mice (332). Nav1.7 expression in Nav1.8-positive thetic fibers, which do not normally innervate DRG neu-
neurons (which includes the majority of nociceptors), there- rons, have been found to be sprouting into the DRG after
fore, appears to be redundant for mediating noxious heat injury. Sprouting into the DRG has been shown to result in
and cold behavior. Nav1.7 expression in a wider population the formation of a dense plexus around individual somata,
of sensory and sympathetic neurons is, however, required described as a “basket” formation. Labeling of the sympa-
for correct noxious processing in a nonredundant fashion. thetic fibers demonstrated sprouting in the DRG peaked 3
Electrophysiological recordings from spinal lamina V wide days post-spinal nerve ligation, which coincided with an
dynamic range neurons (WDR) corroborated these behav- increase in spontaneous activity in large- and medium-di-
ioral results. WDR responses from Nav1.7Advill mice are ameter neurons (488). Approximately 70% of DRG neu-
attenuated in response to noxious heat and mechanical rons that were surrounded by sympathetic nerve baskets
stimuli; however, only mechanically evoked and not heat- exhibited spontaneous activity, usually with bursting activ-
evoked responses are attenuated in Nav1.7Nav1.8 mice ity, compared with only 15% in the total population of
(317). DRG neurons. Furthermore, these neurons were more
likely to express nociceptor markers, such as CGRP, sub-
Interestingly, when assessing noxious behavior using the stance P, and TrkA receptor. Transection of the small dorsal
hotplate test [a behavioral response that requires supraspi- ramus, in addition to the ventral ramus, reduced the levels
nal integration (277)], only Nav1.7Wnt1 mice show attenu- of sprouting after injury and significantly reduced sponta-
ated responses, with Nav1.7Advill mice showing normal be- neous activity (488), suggesting sympathetic innervation is
havior. Chemical sympathectomy in wild-type (WT) mice a vital mediator of enhanced excitability after injury. In
had no effect on behavioral responses on the hotplate, sug- corroboration, deletion of Nav1.7 in all DRG neurons did
gesting sympathetic neurons alone do not determine re- not alter the development of cold or mechanical allodynia,
sponses to the hotplate test. However, when chemical sym- suggesting that, unlike CCI, Nav1.7-positive DRG neurons
pathectomy is combined with Nav1.7 deletion in sensory do not account for hypersensitivity following SNT (316). In
neurons using Nav1.7Advill mice, the hotplate response is contrast, Nav1.7Wnt1 mice do not develop cold (316) or
again attenuated (317). Therefore, Nav1.7 is likely to be mechanical allodynia (317), implying Nav1.7 expression in
required in both sensory and sympathetic neurons for nor- sympathetic neurons is required for SNT-induced allo-
mal responses to supraspinally mediated noxious heat dynia.
avoidance behavior.
Investigations into the role of Nav1.7 in visceral pain mod-
The same approach using the three conditional knockout els have demonstrated little involvement. Nav1.7Nav1.8 mice
mice was used to assess neuropathic pain in the context of showed no significant difference in behavioral assays of
multiple different injury models. CCI (28) of the sciatic acute or chronic visceral pain models. Similarly, the stimu-
nerve results in an inflammatory response with a large cy- lus-response function of visceral afferents was unchanged
tokine involvement (264). After CCI, WT mice develop ro- (220), suggesting Nav1.7 is not involved with the onset or
bust cold and mechanical allodynia; however, Nav1.7Nav1.8 maintenance of chronic visceral pain.
mice do not develop cold allodynia, but do develop mechan-
ical allodynia. Ablation of Nav1.7 in all DRG neurons To investigate possible mechanisms for the analgesia in
(Nav1.7Advill) and in all tissue derived from the neural tube both human patients and mutant mice with Nav1.7 loss-of-
(Nav1.7Wnt1) results in mice with attenuated CCI-induced function mutations, alterations in the transcriptome of
cold and mechanical allodynia. Therefore, Nav1.7 expres- whole DRG after genetic ablation of Nav1.7 were studied.
sion within Nav1.8-positive nociceptors is required for the Conditional ablation of Nav1.7 in sensory neurons resulted in a
development of cold allodynia, but is largely redundant for dramatic alteration in gene expression compared with global de-
the development of mechanical allodynia. Whereas, Nav1.7 letion of Nav1.8 and Nav1.9 (number of genes ⬎1.5-fold dys-

regulated: Nav1.7Advill, 194 genes; Nav1.8Global, 17 genes; littermate controls (317). Concurrently, peripheral input
Nav1.9Global, 64 genes) (318). The levels of Penk mRNA, the into lamina V WDR neurons was unable to induce wind-up
precursor of Leu- and Met-encephalin, were upregulated by (317), a process requiring substance P release from sensory
nearly fourfold in Nav1.7, but not in Nav1.8 or Nav1.9-null mu- neurons (213). The pain behavior related to intraplantar
tant mice (318). Met-enkephalin immunoreactivity was formalin injection comprises an initial phase of nociceptor
correspondingly enhanced by more than twofold in the activation followed by a secondary phase proposed to result
superficial dorsal horn of Nav1.7-null mice, suggesting from activity-dependent sensitization of dorsal horn neu-
enhanced expression in the central terminals of sensory rons (84). Nav1.7Advill mice show almost a 75% reduction
primary afferents (318). These findings led to the hypoth- in phase II of the formalin test, which may be explained by
esis that enkephalin-mediated analgesia contributes to a decreased peripheral input into the CNS, a reduction of
the insensitivity of Nav1.7-null mice and CIP (congenital substance P release, and the related lack of wind-up in CNS
insensitivity to pain) humans. The authors demonstrated dorsal horn neurons.
that blockade of Nav1.7 with TTX at 500 nM (to leave
the TTX-R sodium channels active) enhances Penk ex- Genetic ablation of Nav1.7 and subsequent behavioral
pression, suggesting sodium influx through Nav1.7 analysis has made it very clear that Nav1.7 is vital for acute
downregulates Penk (318). As previously found (317), pain sensation and also contributes to sensitization in a
Nav1.7Advill mice were hyposensitive to both noxious me- number of persistent pain models. We have discussed a
chanical and thermal stimuli. When these mice were ad- number of possible mechanisms by which a lack of Nav1.7
ministered the opioid antagonist naloxone, both thermal causes a defect in nociceptive processing. These include an
and mechanical withdrawal thresholds were restored to upregulation of endogenous opioid signaling, a failure of
littermate control levels (318). In addition, evoked activ- action potential initiation in the peripheral terminals, a fail-
ity of WDR neurons in the deep dorsal horn of ure to transmit action potentials to second-order neurons in
Nav1.7Advill mice, which were reduced compared with the spinal cord (a correlate of its role in the olfactory sys-
littermates, were enhanced on naloxone administration tem), and defective neurotransmitter release by presynaptic
terminals. Analysis of Nav1.7 transgenic mice has actually
in response to mechanical and thermal stimulation.
provided evidence to support all of these actions.
The effect of opioid antagonism was also examined in a
human Nav1.7-null CIP individual [described and geno- B. Nav1.7 Gene Variants and Pain Disorders
typed by Nilsen et al. (334)]. Ordinarily, the individual was
completely unable to detect a radiant heat pulse, delivered 1. Congenital insensitivity to pain
by neodymium:yttrium-aluminum-perovskite (Nd:YAP) la-
ser pulses, which selectively stimulate intra-epidermal free- Case reports in the medical literature of individuals who
nerve endings, providing a nociceptive input without touch. were born unable to experience pain date back over 80 yr
During naloxone application, the probability of detecting (22, 96). This condition was given the term “congenital
the stimulus increased to 80%, and the individual even insensitivity to pain” (CIP), although this is somewhat im-
mentioned pain in one leg that had suffered multiple frac- precise. Pain is defined as a percept, not a stimulus, and so it
tures (318). Recent evidence suggests that, as well as leading is not possible to be sensitive or insensitive to pain but only
to increased antinociceptive signaling through opioids, the the relevant stimulus. These individuals do not feel pain in
absence of Nav1.7 may also result in reduced pronocicep- response to injurious stimuli that would normally evoke
tive G protein-coupled receptor (GPCR)-mediated signaling pain. Another term that has been suggested is congenital
via 5-HT4 receptors within sensory neurons (240). The au- indifference to pain (262). These individuals do not feel
thors suggest that there is a balance between GPCR-medi- pain no matter what the injurious stimulus is (mechanical,
ated pronociceptive signaling and opioid receptor-mediated thermal, or chemical) and no matter where the stimulus is
antinociceptive signaling, which is controlled in part by applied to the body (skin, muscle or viscera). As a result,
Nav1.7. The loss of Nav1.7 shifts the balance toward anti- numerous painless injuries, including fractures, burns, in-
nociception, resulting in lifelong analgesia (240). Future jury to the mouth and tongue, lead to a high level of disabil-
studies will be required to determine the exact mechanism ity. Childhood years can be particularly problematic before
by which Nav1.7 modulates GPCR signaling. individuals learn mechanisms to avoid disabling injury.
Such individuals highlight the critical protective function of
The discovery that postsynaptic responses were virtually acute nociception.
absent in the olfactory glomeruli of conditionally ablated
Nav1.7 mice suggests a role at the presynaptic terminal Early studies noted an inheritance pattern consistent with
(477). When assessing neurotransmitter release in autosomal recessive inheritance. It was noted that there was
Nav1.7Advill mice, electrical stimulation of isolated sciatic remarkable selective deficits in nociception, and that other
nerve roots failed to increase substance P release in the sensory modalities, such as touch, vibration, and proprio-
dorsal horn, whereas a significant increase was detected in ception, were intact. In a subset of cases, this condition is

BENNETT ET AL.
due to the fact that the nociceptive system does not develop were normal, and the only other neurological finding on
adequately, either due to deficient NGF signaling [as a con- examination was that these individuals were anosmic (188).
sequence of mutations in NGF or its receptor trkA (136, As distinct to other forms of CIP, nerve and skin biopsy
239)] or caused by mutations in the transcription factor revealed that unmyelinated and small myelinated fibers are
PRDM12 (75). In all cases, there is virtually absent inner- usually structurally normal (91, 92, 188, 263), although
vation of the epidermis due to presumed developmental
more recent reports suggest that, in some individuals, there
failure or degeneration of nociceptive fibers.
is a partial reduction in intraepidermal nerve fiber density
(IENFD) (FIGURE 7B) (334, 373). The phenotype has re-
In 2006 a number of kindreds were reported with a distinct
clinical phenotype of CIP, the genetic basis of which was cently been extended by the report of two Japanese kindreds
loss-of-function mutations in Nav1.7 (91). These individu- with a homozygous loss-of-function mutation in SCN9A.
als demonstrated a selective deficit in feeling pain in re- In some of these cases, insensitivity to pain did not develop
sponse to noxious stimuli, with other sensory modalities until adolescence. It was associated with evidence of a sen-
being preserved. Cognitive, motor, and autonomic function sory neuropathy on the basis of neurophysiology and a
FIGURE 7. Congenital insensitivity to pain (CIP) mutations. A: Nav1.7 ␣-subunit structure showing the
location of amino acids affected by loss-of-function mutations that are linked to CIP. B: photomicrograph of skin
section immunolabeled for PGP9.5 to show nerve fibers and collagen type IV to show the basement mem-
brane. Fibers can be seen to cross into the epidermis (arrows); however, intraepidermal nerve fiber density
was reduced overall. Scale bar, 50 ␮m. [Adapted with permission from Ramirez et al. (373).]

reduction of myelinated sensory afferents on nerve biopsy (FIGURE 8B), which is exacerbated by warmth and relieved
(503). by cooling. This condition was first described by the Amer-
ican physician Dr. Weir Mitchell in the 19th century (319).
Homozygosity mapping in consanguineous kindreds fol- The condition principally affects the feet, but may also in-
lowed by candidate gene sequencing revealed bi-allelic-null volve the hands and infrequently the nose and the ears.
mutations in Nav1.7 (91) (see TABLE 1 and FIGURE 7A, for Episodes of pain and erythema are triggered by warmth,
reported mutations to date). These mutations most com- exercise, prolonged standing, and sometimes by alcohol.
monly would be predicted to result in truncated (and non- Patients often find it difficult to wear enclosed shoes and,
functional) protein, although more recently a number of because pain is classically relieved by cooling (130, 421),
missense mutations have been described. In the initial re- are at risk of developing skin ulceration due to prolonged
port, heterologous expression of these mutant channels and standing in cold water. Brain perfusion data found that
electrophysiological analysis suggested that these mutations significant changes in cerebral blood flow generated by the
would result in complete failure in the ability of these chan- contrast of baseline pain vs. blocks of cooling relief oc-
nels to carry current (91), and a subsequent publication curred in many brain regions involved in processing acute,
suggesting impaired surface expression of mutant Nav1.7 phasic, and tonic pain (FIGURE 8C) (421). Some patients
(92). Interestingly, more recently it has been noted that develop ongoing pain (and are virtually never pain free)
some CIP-associated missense mutations can, in fact, form with superimposed fluctuations. EM can be secondary to
functional channels, although channel function is impaired blood dyscrasias, connective tissue disorders, and drug re-
with reduced peak currents and a depolarizing shift in the action; however, it can also be inherited.
voltage dependence of activation (139). It is interesting to
speculate how much residual channel function would be Inherited EM (IEM) usually presents in the first 2 decades of
required for an individual to feel pain, given that patients life; however, presentations at birth (421) and as late as the
carrying heterozygous loss-of-function mutations in Nav1.7 6th decade of life (95) have been reported. In 2004, Yang et
have normal pain sensibility. Interestingly, one patient with al. (496) reported a Chinese pedigree suffering from EM
CIP who has compound heterozygote mutations in Nav1.7 with an autosomal dominant pattern of inheritance in
(including one in the COOH-terminal domain) never expe-
which linkage analysis and candidate gene sequencing re-
rienced pain, but did have some retained nociception in that
vealed a point mutation in Nav1.7 (L858H); sequencing of
he notes a high-frequency “tingling” sensation to high-
further probands revealed another mutation: I848T. Mul-
threshold noxious stimuli (373).
tiple missense mutations in Nav1.7 have now been associ-
ated with IEM, and these are summarized in TABLE 2 and
The physiological mechanism by which such individuals do
FIGURE 8A. There is not always a family history, as de novo
not feel pain has still not been fully clarified (see sect. IIIA6,
mutations have been described (200).
Examination of Nav1.7 function in pain signaling using
rodent models, for a full discussion in relation to mutant
Microneurographic recordings from sensory axons in pa-
mice). It has been reported by a number of authors that the
tients suffering from EM (some of whom had confirmed
pruritic agent histamine can evoke a local neurogenic flare
mutations in Nav1.7) have shown that mechanically insen-
in these individuals, which requires short-range action po-
tential propagation in sensory terminals (92). We have also sitive nociceptors have a pathologically high rate of spon-
found that algogens can evoke a flare in other CIP individ- taneous activity (329, 346), directly confirming nociceptor
uals; however, this is never associated with pain (DLB, un- hyperexcitability as the basis of neuropathic pain in IEM.
published observations). This would suggest that nocicep- The I848T mutation was associated with supranormal con-
tive afferents lacking Nav1.7 are not entirely nonfunctional, duction in velocity recovery cycle assessment in nociceptors
and at least some ability to transduce noxious stimuli is but not sympathetic axons (329). Many Nav1.7 mutants
preserved. This also correlates with the fact that, in mice have now undergone electrophysiological analysis at a cel-
with absent Nav1.7, action potentials can be recorded in lular level, allowing comparison of the nature/site of muta-
nociceptive afferents after suprathreshold stimuli, although tion in Nav1.7 with channel function and clinical phenotype
firing frequency is reduced (183). The function of Nav1.7 (see TABLE 2). A number of pathophysiological mechanisms
may not therefore be confined to regulating the excitability appear to lead to enhanced excitability, and key pathophys-
of peripheral nociceptor terminals. It has also been found to iological mechanisms are a hyperpolarizing shift in the volt-
be important for ingress of action potentials into the central age dependence of activation and increase discharge on
terminals of nociceptors and neurotransmitter release slow depolarization (ramp currents) (102, 121). When mu-
within sensory neurons (240). tant channels are expressed in DRG neurons, it results in a
lowered threshold for the generation of action potentials
2. Inherited erythromelalgia and a higher frequency of repetitive firing. Dynamic clamp
studies enable a conductance to be titrated to “physiologi-
Erythromelalgia (EM) is a disabling chronic pain condition cal” levels, for instance in the case calibrating a 1:1 ratio of
characterized by pain and erythema of the extremities WT and mutant channel, given that IEM-related mutations

1098
TABLE 1. Summary of published mutations linked to congenital insensitivity to pain showing the clinical presentation and electrophysiological findings
PubMed Absent Intact Large Autonomic Nerve Nerve
Mutations ID Mutation Anosmia? Pain? Fibers? QST Function Flare Response IENFD Biopsy Conduction
F258fs; R830X 19304393 Compound Y Y Y Warm Normal NT Reduced NT Normal motor

heterozygous reduced, reduced
pain absent amplitude
SAPs
R277X 17470132 Homozygous NT Y Y NT Y NT NT Normal Normal motor
and sensory
Y328X 17470132 Homozygous NT Y Y NT N Histamine normal NT Normal Normal motor
and sensory
Y328X 17597096 Homozygous NT Y Y NT NT NT NT NT NT
K376Q; G375Afs 24188911 Compound Y Y Y NT Normal sweat, and Histamine NT NT Normal motor
heterozygous sympathetic skin present but and sensory
response reduced
S459X 17167479 Homozygous NT Y Y NT NT NT NT Normal Normal motor
and sensory
R523X; 23129781 Compound NT Y NT NT N NT Normal Normal Normal motor
IVS8–2A⬎G heterozygous and sensory
R523X 20628234 Homozygous N Y Y NT NT NT NT NT NT
I767X 17167479 Homozygous NT Y Y NT NT NT NT Normal Normal motor
and sensory
R830X; E1773fs 25253744 Compound Y Y Y Warm/cool NT NT Reduced NT Normal motor
heterozygous reduced, and sensory
pain absent
R830X 17470132 Homozygous NT Y Y NT Normal NT NT Normal NT
BENNETT ET AL.
R896Q 20635406 Homozygous NT Y Y NT NT Histamine normal NT NT Normal motor

borderline
reduction
SAPs
W897X 17167479 Homozygous NT Y Y NT NT NT NT NT NT
W897X; G1725R 26292973 Compound Y Y Y Warm NT NT NT NT Normal motor
heterozygous reduced, and sensory
pain absent
E919X 25309764 Homozygous Y Y N NT NT NT NT NT Reduced
SAPs.
Charcot
joints
Physiol Rev • VOL 99 • APRIL 2019 • www.prv.org

E970X 24813348 Homozygous NT Y Y NT Reduced sweating NT NT NT Normal motor
and sensory
F1200LfsX33 17470132 Homozygous NT Y Y NT Normal Histamine normal NT Normal NT

M1190X 27747863 Homozygous N Y NT NT NT NT NT NT NT
A1236E; L1831X 25995458 Compound Y Y NT NT NT NT NT NT NT
heterozygote
L1331F 23596073 Homozygous Y Y Y NT Impaired sweating NT NT Reduced Normal motor
myelinated and SAPs
axons
Continued
are inherited in a heterozygous fashion. This has been used

to investigate the effect of WT Nav1.7 and the L858H mu-
Normal motor
Normal motor
conduction
amplitudes
Conduction
tation. There was a linear correlation between the level of
reduced
Nerve
sensory
velocity
normal
SAPs
Slowed
Nav1.7 conductance and the lowered current threshold in
and
and
NT
NT
NT
DRG neurons. The L858H mutation increased channel ac-
tivity at membrane potentials below the action potential
threshold, producing a large, persistent current that depo-
Biopsy
Nerve
larized the membrane potential as well as increasing the

Normal
Normal
Normal
current at interspike intervals (466).
NT
NT
NT
There is a relationship between biophysical abnormalities
IENFD
Normal
evoked by channel mutations and the clinical severity in pa-

NT
NT
NT
NT
NT
tients with these mutations. Those mutations producing a
IENFD, intraepidermal nerve fiber density; N, no; NT, not tested; QST, quantitative sensory testing; SAPs, sensory action potentials; Y, yes.
larger hyperpolarizing shift in the voltage dependence of acti-
Histamine normal
Flare Response
vation are associated with a younger age of onset (95, 196).

There are “hot spots” for mutations, for instance in the linker
between transmembrane segment S4 and S5 (FIGURE 8A). This
NT
NT
NT
NT
NT
region connects the voltage sensor (segments S1–S4) to the

channel pore (S5 and S6) (497), so mutations in this region
may be expected to have an impact on channel activation
Autonomic
Function
properties. One such mutation in this hotspot (L858F) did not

affect peak current densities (FIGURE 8, D AND E), but shifted
Normal
Normal
Normal
Normal
voltage-dependent activation in a hyperpolarized direction

NT
NT
(FIGURE 8F). Furthermore, deactivation was markedly slower

Table 1.—Continued
for the L858F variant at depolarized potentials, which could

Cool reduced
allow more channels to be open and increase the probability of

QST
a ramp current (FIGURE 8G) (200). Studying the effect of

Nav1.7 mutations has also given insight into the clinical ob-
NT
NT
NT
NT
NT
servation that warmth triggers pain: DRG neurons expressing

mutant Nav1.7 channels from IEM patients increase firing in
Intact Large
Fibers?
response to a warm stimulus compared with neurons express-

NT
NT
Y
Y
Y
Y
ing WT channels (182). Another important feature of the ef-

fects of mutant Nav1.7 is that, at a cellular level, excitability
changes depend on the complement of other VGSCs ex-
Absent
Pain?
pressed. This was demonstrated when the L858H mutant was

Y
Y
Y
Y
Y
expressed in DRG vs. sympathetic ganglia (199, 400). This

mutation leads to gain of function in Nav1.7, which facilitates
Anosmia?
activation of Nav1.8 (which has a more depolarized voltage

NT
NT
NT
NT
dependence of activation than Nav1.7), leading to hyperexcit-

Y
ability. Sympathetic neurons, however, which do not express

Nav1.8, became hypoexcitable in the presence of the same
heterozygous
heterozygous
heterozygous
heterozygote
mutation based on depolarization-induced inactivation of

Mutation
Homozygous
Homozygous
Compound
Compound
Compound
Compound
Nav1.7.
Pain associated with IEM can be extremely difficult to treat,

and there is very little evidence base on which to guide
20635406
17470132
17470132
17470132
20692858
25995458
treatment. Drugs such as anticonvulsants and local anes-

PubMed
ID
thetics, which have broad activity against VGSCs, are often

used. Dosing is often limited by cardiac and CNS side ef-
fects; furthermore, response (both at a clinical and electro-
R1370_L1374del;
physiological level) is dependent on the exact mutation.

C1719R; c.3467
W1775R; c.901
Mutations
I1493SfsX8
Carbamazepine and mexiletine have shown efficacy in the

I1235fsx2
⫹ 3 delA
⫹ 5G⬎C
context of certain mutations (82, 94, 495). A recent ad-

W1689X
K1659X;
R1488X
vance has been the use of structural modeling to predict

pharmacological responsiveness (495). IEM patients with
the V400M Nav1.7 mutation showed a clinical response to

BENNETT ET AL.
carbamazepine, which also reversed the hyperpolarizing to predict pharmacological responsiveness, and indeed the
shift in the voltage dependence of activation of the mutant S241T mutation was shown to be responsive to carbamaz-
channel (166). Structural modeling revealed that a distinct epine in vitro; two patients with the S241T variant reported
IEM mutation (S241T) was only 2.4-Å apart from V400M less pain following carbamazepine treatment in a placebo-
in the folded Nav1.7 channel. Mutant cycle analysis dem- controlled, double-blind study and also showed reduced
onstrates that these mutations were energetically coupled activation of pain-associated areas using functional brain
during channel activation. This information was then used imaging (182).

A number of companies are attempting to develop selective attacks with apnea and bradycardia. Tonic attacks may
blockers of Nav1.7, and patients with IEM are being re- initially be confused with epilepsy; however, electro-en-
cruited to proof-of-concept trials (189). The advent of tech- cephalogram is normal. As well as physical factors,
nology to generate human-induced pluripotent stem cells strong emotion can also be a trigger for attacks. Between
and their differentiation into nociceptors (70) has made it episodes, physical and neurological examination is nor-
possible to directly study the impact of ion channel muta- mal. Most patients present at birth or in infancy, and the
tions on human sensory neurons in vitro relating this to frequency of rectal pain attacks often decreases with age
treatment response. Cao et al. (63) generated induced plu- (although the frequency of ocular and mandibular pain
ripotent stem cells (iPSCs) from four IEM patients with four may not decrease with age). Linkage analysis followed by
distinct Nav1.7 mutations. Nociceptors differentiated from candidate gene sequencing identified missense mutations
these iPSCs demonstrated evidence of hyperexcitability in Nav1.7 segregating with this disorder (161) (TABLE 3
with a higher incidence of spontaneous activity and lowered and FIGURE 9 summarize Nav1.7 mutations that have
rheobase. Warming of these neurons reduced rheobase fur- been associated with PEPD). The effects of mutations
ther in the IEM-derived lines, but not in control. These associated with PEPD are distinct from those seen in IEM
changes could be largely reversed by a selective blocker of in that they lead to impaired fast inactivation of the
Nav1.7. There was a trend toward a correlation between the channel, resulting in a persistent sodium current (161), as
degree of hyperexcitability in vitro and the severity of the well as more rapid recovery from fast inactivation (119).
clinical phenotype. Most subjects treated with a selective Many of these mutations are located within or close to
blocker of Nav1.7 (as part of a randomized, double-blind, the highly conserved DIII-DIV inactivation particle
placebo-controlled, cross-over study in a small cohort of (IFMT), which is known to be critical for VGSC inacti-
patients) reported reduced heat-induced pain. This study vation (479), consistent with the functional impact of
shows how interesting parallels can be drawn between clin- these mutations. A more recently recognized feature
ical phenotype and electrophysiological outcomes in hu- of these mutations is that they can lead to the generation
man cellular models. of resurgent currents (243, 453). These are unusual cur-
rents: under normal circumstances after opening, VGSCs
3. Paroxysmal extreme pain disorder will then rapidly inactivate. Inactivated channels cannot
reopen until they have been hyperpolarized for many
Paroxysmal extreme pain disorder (PEPD) was originally milliseconds, and this is the basis of the refractory period.
known as “familial rectal pain.” Hayden and Grossman In contrast to this dogma, channels may reopen during
(210) and subsequently Dugan (133) described key aspects the repolarization of the membrane potential and gener-
of the condition, including severe pain centered on the rec- ate resurgent currents. Such currents could contribute to
tum/sacral area, triggered by mechanical stimulation and spontaneous and high-frequency firing. One theory as to
associated with a flare in the sacral region and thigh. Some their origin is that there is an intracellular blocking factor
patients also described ocular and/or mandibular pain. that binds to open VGSCs, preventing classical VGSC
Both authors reported this condition affecting multigenera- inactivation. It then unbinds during repolarization, gen-
tional family pedigrees, which demonstrated an autosomal erating the resurgent current. These currents are ob-
dominant pattern of inheritance. The widespread nature of served as a consequence of Nav1.7 mutations associated
the pain and concern expressed by patient groups led to the with PEPD, but not IEM (453). Computer simulations
condition being renamed as “paroxysmal extreme pain dis- indicated that resurgent currents associated with Nav1.7
order” (162, 163). Pain is also associated with autonomic mutation could induce high-frequency action potential
features, which may include flushing, which can result in firing in nociceptive neurons and so contribute to hyper-
harlequin color change, lacrimation, rhinorrhea, and tonic excitability in PEPD (453).
FIGURE 8. Inherited erythromelalgia mutations (IEM) and electrophysiological characteristics. A: Nav1.7 ␣-subunit structure showing the
location of amino acids affected by gain-of-function mutations that are linked to IEM. B: photograph of an IEM patient’s legs showing erythema
up to the level of the midcalf. Patient is attempting to relieve pain by cooling feet in cold water. C: brain perfusion data from a single IEM patient
case study acquired with the multi-TI whole brain pseudo-continuous arterial spin labeling sequence. Perfusion maps represent the contrast of
the subject’s erythromelalgia-associated pain vs. blocks of cooling pain relief. Significant changes in cerebral blood flow occurred in many regions
shown previously to be involved in processing acute, phasic, and tonic pain, including the following: precentral gyrus (I), postcentral gyrus (J),
anterior cingulate cortex (K), middle cingulate cortex (L), posterior cingulate cortex (M), and angular gyrus (N). E and F: representative currents
produced by cells expressing wild-type (D) or mutant L858F channels (E), elicited using a range of depolarizations (⫺80 mV to ⫹40 mV) from
a holding potential of ⫺120 mV. L858F variant is highlighted in red in A. F: activation and steady-state inactivation curves (using a series of
prepulses, from ⫺150 mV to 0 mV for 500 ms before a test pulse to 20 mV) for wild-type and mutant L858F channels. Note the hyperpolarizing
shift in activation and the depolarizing shift in inactivation for the mutant channel. G: deactivation time constants (after activation by pulses to
20 mV for 0.5 ms), plotted as a function of deactivation potential for wild-type and L858F mutant channels. Note the markedly slower
deactivation for the mutant channels at potentials more depolarized than ⫺70 mV. [Reproduced with permission from Bennett and Woods (27)
(B), Segerdahl et al. (421) (C), and Han et al. (200) (D–G).]

1102
TABLE 2. Summary of published mutations linked to inherited erythromelalgia showing the electrophysiological characteristics
Suprathreshold
Slow Persistent Spontaneous Current Response to
Amino Acid PubMed ID Nucleotide V1/2 act V1/2 inact Repriming Inactivation Deactivation Resurgent Ramp Current Activity RMP Threshold Current Injection
Q10R 19369487 29A⬎G Hyperpol UC UC Enhanced Slower NT UC No NT UC Reduced Increased

I136V 18171466 406A⬎G Hyperpol UC Faster Enhanced Slower NT Increased No NT NT NT NT
I136V 23383113 406A⬎G Hyperpol Depol Faster NT NT NT NT No NT NT NT NT
S211P 20123784 631T⬎C Hyperpol UC UC Enhanced Slower NT Increased No NT NT NT NT
F216S 16988069 647T⬎C Hyperpol UC UC Enhanced Slower NT Increased No NT NT NT NT
I234T 20385509 701T⬎C Hyperpol UC NT Enhanced Slower NT Increased NT NT NT NT NT
S241T 17008310 721T⬎A Hyperpol UC NT Enhanced Slower NT Increased No NT NC Reduced Increased
23149731
L245V 25995458 733C⬎G UC UC NT Enhanced Slower UC Increased Yes NT NT NT NT
N395K 17430993 1185C⬎A Hyperpol UC NT Impaired Slower NT NT No NT NT NT NT
V400M 19557861 1198G⬎A Hyperpol Depol NT UC Slower NT Increased No NT NT NT NT
G616R 20478850 1846G⬎A UC Depol NT NT NT NT NT No Increased Depol Reduced Increased
L823R 19800314 2468T⬎G Hyperpol Hyperpol NT NT Slower NT NT No NT NT NT NT
I848T 15385606 2543T⬎C Hyperpol UC NT Impaired Slower No Increased No NT Depol Reduced Increased
19369487
21788423
I848T 23383113 2543T⬎C Hyperpol Depol Faster NT NT NT NT No NT NT NT NT
G856R 28381558 2566G⬎C Hyperpol UC Faster Enhanced Slower NT UC No NT NT NT NT
G856D 22286749 2567G⬎A Hyperpol Depol UC Enhanced Slower NT Increased Yes Increased Depol Reduced Increased
BENNETT ET AL.
L858H 15385606 2573T⬎A Hyperpol UC NT Enhanced Slower No Increased No NT Depol Reduced Increased
16702558
21788423
L858F 16392115 c2572C⬎T Hyperpol Depol Faster UC Slower NT Increased No NT NT NT NT
A863P 17135418 2587G⬎C Hyperpol Depol Faster Impaired Slower NT Increased No NT Depol Reduced Increased
V872G 19162012 2616T⬎G Hyperpol UC NT Slower NT Increased No NT NT NT NT
Q875E 25575597 2623C⬎G Hyperpol UC NT Enhanced Slower NT NT NT NT NT NT NT
L955Del 21705421 c.2863_ Hyperpol UC NT Enhanced NT NT Increased Yes NT UC Reduced Increased
2865del TTA

P1308L 20429905 3971C⬎T Hyperpol UC UC UC Slower NT Increased No NT UC Reduced Increased
V1316A 23376079 3947T⬎C Hyperpol UC NT Enhanced NT NT Increased No NT NT NT NT
V1316A 23383113 3947T⬎C Hyperpol Depol Faster NT NT NT NT No NT NT NT NT

F1449V 15958509 4393 t ⬎ G Hyperpol Depol Faster UC UC NT UC No NT UC Reduced Increased
W1538R 23292638 4612T⬎C Hyperpol UC UC UC NT NT NT No NT NT NT NT
A1632T 24311784 4894G⬎A UC Depol NT UC Enhanced UC NT No Increased UC Reduced Increased
A1632G 27413160 4895C⬎G Hyperpol Depol NT UC NT NT NT No Increased Depol Reduced Increased
A1746G 23292638 5237C⬎G Hyperpol UC Faster Enhanced NT NT NT No NT NT NT NT
Depol, depolarized; Hyperpol, hyperpolarized; NT, not tested; RMP, resting membrane potential; UC, unchanged; V1/2 act and V1/2 inact, half-maximal voltage activation and
inactivation, respectively.
Carbamazepine is an effective treatment at reducing the
frequency and severity of pain attacks in PEPD and has also
Suprathreshold
Response to been shown to reduce persistent sodium current generated
Increased
Increased
Increased
Injection
Current by PEPD-associated mutant Nav1.7 (161). This is consis-
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
tent with the effect of carbamazepine in stabilizing the in-
active state of VGSCs (419) in a use-dependent manner,
inactivation, respectively. *M1627K–V1/2 activation is unchanged when no peptide present, but is significantly depolarized when the NavB4 peptide is coexpressed.
which can reduce repetitive firing.
RMP Threshold
Reduced
Reduced
Current
In general, Nav1.7 mutations causing IEM result in en-

UC
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
hanced channel activation, and PEPD mutations cause im-
paired inactivation (and are also associated with persistent
UC
UC
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
TABLE 3. Summary of published mutations linked to PEPD showing the electrophysiological characteristics
and resurgent currents); there are notable exceptions to this

Persistent Spontaneous
rule, however. For instance, the G616R (78), A1632T

Activity
(135), and L245V (139) mutations are associated with

NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
IEM. These mutations did not have an effect on channel
activation, but were associated with a depolarizing shift in
fast inactivation of Nav1.7 (in the case of G616R, this was
Increased
Current?
specific to the adult long splice variant of the channel). The

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
NT
NT
NT
No
No
L245V mutation even resulted in persistent currents due to

incomplete fast inactivation (139), whereas formerly these
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Increased
currents were thought to be specific to PEPD. Furthermore,

Ramp
although IEM and PEPD are usually clinically distinct, cases

NT
NT
NT
NT
NT
have been reported demonstrating clinical features of both

disorders; the associated A132E mutation had characteris-
Repriming Inactivation Deactivation Resurgent
tics common to PEPD and IEM (146). In summary, al-

Yes
Yes
Yes
Yes
NT
NT
NT
NT
NT
NT
NT
NT
NT
though there are some general principles regarding the re-

lationship between the clinical phenotypes of IEM and
PEPD and the biophysical changes in channel function,
Slower
these are not absolute. There is a further unanswered ques-

UC
UC
UC
UC
UC
NT
NT
NT
NT
NT
NT
NT
tion, given that Nav1.7 is expressed at the soma, the axon,

and the terminals, why do IEM and PEPD have such distinct
anatomical distributions affecting the extremities and prox-
Impaired
Impaired
Impaired
Impaired
Impaired
Impaired
imal regions, respectively? This may relate to as yet undis-

Slow
covered protein interactors or trafficking of these ion chan-

UC
UC
NT
NT
NT
NT
NT
nels.
Faster
Faster
Faster
Faster
Faster
Faster
Faster
4. Small-fiber neuropathy and painful diabetic

NT
NT
NT
NT
NT
NT
neuropathy
Depol⫹⫹
Depol⫹⫹
In the condition of small-fiber neuropathy (SFN), the axons

inact
V1/2
Depol
Depol
Depol
Depol
Depol
Depol
Depol
Hyperpol Depol
Depol
Depol
Hyperpol Depol
of unmyelinated and thin myelinated degenerate usually in

a length-dependent manner (225, 454). Patients will typi-
cally present with burning pain of the feet. Although pa-
V1/2
act
Depol
Depol
Depol
Depol
Depol
Depol
UC*
tients may note altered temperature sensibility, unlike IEM,

UC
UC
UC
UC
the symptoms are not usually exacerbated by warming and

Amino Acid PubMed ID Nucleotide
relieved by cooling. Erythema is present in some cases of

4819G⬎C
4895C⬎A
3892G⬎T
3892G⬎T
3895G⬎T
4879T⬎A
4879T⬎A
4879T⬎A
4383T⬎C
4383T⬎C
4391C⬎T
4391C⬎T
4835T⬎C
SFN, but it is relatively mild. By definition, large-fiber sen-

sory function is not impaired. On examination, patients
usually have loss of the ability to feel pin prick as painful in
18599537
20429905
18599537
21115638
18599537
17145499
17145499
21115638
21079636
25285947
18803825
17145499
21115638
18945915
the feet and may show impairments in detecting warmth

and cool; however, light touch, vibration, proprioception,
and deep tendon reflexes are normal. Some patients also
demonstrate significant autonomic involvement.
M1627K
M1627K
M1627K
G1607R
A1632E
L1612P
V1298F
V1298F
V1299F
I1461T
I1461T
T1464I
T1464I
Diagnosis relies on typical symptoms and signs of small-

fiber function and supportive diagnostic tests showing
small-fiber injury. Normal nerve conduction studies inter-

BENNETT ET AL.
FIGURE 9. Paroxysmal extreme pain disorder (PEPD) mutations . Nav1.7 ␣-subunit structure shows the
location of amino acids affected by gain-of-function mutations that are linked to PEPD.
rogate large-fiber function and are normal. Probably the All of the reported variants result in substitution of highly
best diagnostic test is the demonstration of reduced IENFD conserved amino acids within Nav1.7; it should be noted
assessed using skin biopsy (FIGURE 10, B AND C) (113). The that these variants are distinct from those causing IEM and
new modality of corneal confocal microscopy enables the PEPD (Nav1.7 variants associated with SFN are illustrated
noninvasive visualization of small fibers (451). In addi- in TABLE 4 and FIGURE 10A). Electrophysiological analysis
tion, quantitative sensory testing may reveal elevated has shown depolarized slow and fast inactivation and in-
thermal thresholds, and autonomic function tests may be creased resurgent currents, but did not show the hyperpo-
abnormal. larizing shift in the voltage dependence of activation re-
ported in IEM (150, 198). These changes result in DRG cell
SFN may be acquired, for instance, secondary to diabetes hyperexcitability, including a depolarizing shift in resting
mellitus, chemotherapy, or immune dysfunction. A signifi- membrane potential (RMP), reduced current threshold, en-
cant proportion (~50%) has been described as idiopathic. hanced firing frequency, and spontaneous activity.
Gain of function missense variants in Nav1.7 were recently
found in up to 30% of SFN patients in which no underlying Of course, one key issue is how these variants lead to loss
cause had been found (150). Genetic counselling in this of the structural integrity of sensory axons. The I228M
context is difficult, as they are best described as variants Nav1.7 variant, which is associated with SFN, has been
rather than mutations, as some of the reported variants shown to significantly reduce neurite outgrowth in DRG
occur with a minor allele frequency of up to 3.7% (e.g., neurons (FIGURE 10, D–F) (360), which could be pre-
M932L) and the penetrance of these variants are not yet vented by carbamazepine. Enhanced activity of Nav1.7 as
known: Of the eight patients in the initial report, three a consequence of a pathogenic mutation would be pre-
reported similar complaints in family members. Given the dicted to lead to increased sodium load, reversal of the
frequency of these variants, SFN is likely to be distinct to sodium-calcium exchanger (NCX) and increased intra-
IEM and PEPD, which appear to be highly penetrant, and it axonal calcium, which could lead to axonal degenera-
may be that there is an interaction between the variant and tion. In support of such a hypothesis, inhibition of re-
an environmental trigger. The complexity of genetic coun- verse operation of NCX could enhance neurite out-
selling was pointed out in a recent review (476), which growth of DRG neurons transfected with I228M. Similar
emphasized the importance of functional analysis of vari- results have recently been shown in association with the
ants. Better understanding of the structure of Nav1.7 and G856D mutation in Nav1.7, which also leads to DRG cell
how mutations may disturb such structure is also likely to hyperexcitability and was originally found in a pedigree
help in the assessment of whether mutations are pathogenic with SFN, dysautonomia, and acromelia (226). Expres-
(235). sion of G856D Nav1.7 in DRG neurons was noted to be


BENNETT ET AL.
associated with increased intracellular Na⫹ and Ca2⫹ sessed in people with sciatica, amputees with phantom limb
following neuronal depolarization with extracellular K⫹ pain, and patients with post-discectomy lumbar root pain
(148). The combination of metabolic stress and depolar- (377). In addition, in both clinical and experimental pain
ization led to axon degeneration in G856N Nav1.7 ex- studies, the level of pain felt was correlated with the geno-
pressing neurons. This axon degeneration could be pre- type, with the least pain with the WT GG genotype, inter-
vented by inhibition of NCX. These findings are exciting, mediate pain in the GA genotype, and the most pain in the
as they suggest novel treatment targets, which may not AA genotype, suggesting an allelic dose dependency. To
only ameliorate pain, but could be disease modifying and examine the functional effects of the rs6746030 SNPs, the
prevent degeneration and at best promote neural repair two alleles that alter the coding sequence of Nav1.7, result-
in SFN. ing in either 1150R or 1150W (encoded by the minor A
allele), were separately transfected into HEK-293 cells. The
A recent study investigated Nav1.7 variants in a deeply rarer A allele was found to impair slow inactivation at volt-
phenotyped cohort of patients with painful and painless ages close to RMP, which is predicted to increase Nav1.7
diabetic neuropathy. Interestingly, 12 rare Nav1.7 variants activity (377). In a different study, 1150W induced a depo-
were present in 10 out of 111 study participants with pain- larizing shift in the voltage dependence of activation com-
ful diabetic neuropathy, and no rare variants were present pared with 1150R, inducing hyperexcitability in small
in 78 participants with painless diabetic neuropathy. Five of DRG neurons (147), consistent with the clinical phenotype
the rare variants had previously been described in the con- described in Reimann et al. (377). Data described here sug-
text of other neuropathic pain disorders (principally SFN) gest that SNPs in SCN9A resulting in genetic variants of
and seven were novel. Electrophysiological characterization Nav1.7 may alter human pain threshold levels; and this
of two of these variants (M1852T and T1596I) demon- variant may contribute to enhanced pain in common con-
strated gain-of-function changes as a consequence of mark- ditions such as back pain and osteoarthritis.
edly impaired channel fast inactivation (54). There was also
an interesting relationship between sensory phenotype and
genotype: the presence of rare Nav1.7 variants in the con- C. Targeting Nav1.7 Therapeutically to
text of painful diabetic neuropathy was associated with Treat Pain
enhanced sensitivity to pressure pain stimuli.
Given the key role of Nav1.7 in pain perception revealed by
5. Polymorphisms in Nav1.7 and their role in pain the preclinical studies and human genetics, there have been
sensitivity sustained efforts to develop selective inhibitors of this ion
channel. The relatively specific expression of Nav1.7 prin-
High-impact mutations in Nav1.7 have marked effects on cipally in the PNS (with the exception of olfactory neurons)
channel function and clinical phenotype, as described in the provides hope that such analgesics would have not only
previous sections. Single nucleotide polymorphisms (SNPs) efficacy, but also fewer side effects than those currently
in the SCN9A gene have more subtle effects than these available. Therapeutics such as mexiletine, lidocaine, and
major mutations and may act to influence an individual’s carbamazepine are nonselective VGSC inhibitors and are
pain perception. A common polymorphism (rs6746030) currently used as analgesics; these have even shown efficacy
was found in the intracellular loop between domains II and in certain pain channelopathies associated with Nav1.7 mu-
III, with the minor allele occurring at a frequency of ~10%. tations (see above); however, activity against cardiac and
The polymorphism results in an arginine-to-tryptophan brain VGSCs can be dose limiting. A number of different
amino acid change at position 1150, which is an evolution- approaches are being used in the development of Nav1.7
arily conserved residue, suggesting it could be important in inhibitors, including the use of monoclonal antibodies di-
the functioning of the sodium channel. The rarer A allele rected against this channel, peptide toxins, and small mol-
was associated with increased pain perception when as- ecules.
FIGURE 10. Small-fiber neuropathy is associated with reduced intraepidermal nerve fiber density. A: Nav1.7 ␣-subunit structure showing the
location of amino acids affected by gain-of-function mutations that are linked to small-fiber neuropathy. Immunohistochemical staining of skin from
the lower leg in a healthy control (B) and small-fiber neuropathy patient (C) is shown. Number of fibers penetrating into the epidermis
(arrowheads) is greatly reduced in the patient. Dermal/epidermal boundary is illustrated with a dotted line. D: large-field montage image
consisting of a 10 ⫻ 10 field-of-view montage image of a dorsal root ganglion culture 3 days after transfection with Nav1.7 wild-type (WT) ⫹
green fluorescent protein (GFP) constructs, with GFP signal in white. Dahsed lines distinguish individual field-of-view captures. Scale bar ⫽ 1,000
␮M. E: increased magnification of individual neurons transfected with Nav1.7 WT, I228M, ML/VL, and I720K constructs demonstrates
reduced neurite length of I228M-transfected neuron compared with WT. Scale bar ⫽ 250 ␮M. F: quantifications of the total neurite
length/neuron calculated from large-field images and averaged for each condition. Pairwise comparisons between neurites from neurons
expressing WT channels and channel variants I228M, ML/VL, and I720K are presented. Data are normalized to WT values and presented as
means ⫾ SE. *P ⬍ 0.05. [D–F were adapted with permission from Persson et al. (360).]

1. Toxins
Depol, depolarized; NT, not tested; RMP, resting membrane potential; UC, unchanged; V1/2 act and V1/2 inact, half-maximal voltage activation and inactivation, respectively.
Suprathreshold
Response
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Injection
Current
Natural toxins have long been used to probe the function of
to
ion channels (253). There are 12 families of spider venom
NT
NT
peptides that target VGSCs (termed “NaSpTx” families
RMP Threshold
Current 1–12); they consist of 33–35 aa peptides with three disulfide
bridges that form an inhibitory cysteine knot motif.
Reduced
Depol Reduced
Depol Reduced
Depol Reduced
Depol Reduced
Depol Reduced
ProTx-II (isolated from the tarantula Thrixopelma pru-
TABLE 4. Summary of published mutations linked to small-fiber neuropathy showing the electrophysiological characteristics
Depol UC
NT
NT
riens) and huwentoxin-IV (HWTX-IV, Chinese bird spider
Selenocosmia huwena) are two such toxins (314, 356),
UC
NT
NT
which both show selectivity in inhibiting Nav1.7 over other
␣-subunits of VGSCs (416, 437, 486). These toxins target
Activity
the voltage sensor of Nav1.7 (438, 486). They bind to the

Increased
Increased
Increased
Increased
Increased
Increased
Increased S3b-S4 helix-turn-helix (termed the paddle motif) in the

NT
NT
domain II voltage sensor (486). This so-called paddle motif
is targeted by a variety of animal toxins and is important for
Increased
Current?
voltage sensing (11, 55) as it moves in response to changes

Yes
NT
NT
No
No
No
No
No
No
*M932L;V991L is not fully penetrant. It is present in 23% of Latino alleles and 6% of East Asian alleles (EXAC database). in membrane potential: its motion is coupled to pore open-
ing, closing, and inactivation (termed gating) (247). These
Increased TTX-S
toxins, therefore, trap this voltage sensor of Nav1.7 (438,

Ramp
486) in closed configuration, thus inhibiting activation of

Increased
Increased
Increased
the ion channel. Although they both bind to the paddle

motif of domain 2, specific residues within this motif play
UC
UC
UC
UC
UC
substantially different roles in determining the affinities of

inact Repriming Inactivation Deactivation Resurgent
Increased
Increased
these toxins to hNav1.7; ProTx-II (but not HWTX-IV) also

appears to have additional interactions with voltage sensors
UC
NT
NT
NT
NT
NT
NT
of domains I and IV (55, 487). A further peptide “␮-ther-

aphotoxin-Pn3a,” isolated from venom of the tarantula
Accelerated
Pamphobeteus nigricolor was recently reported to be a

highly selective inhibitor of Nav1.7 by binding to domains II
UC
UC
UC
UC
NT
NT
NT
NT
and IV (109).
Slow
Impaired
Impaired
Impaired
Impaired
Impaired
In terms of testing physiological effects of these toxins,

ProTx-II has been shown to block the C-fiber compound
UC
UC
UC
UC
action potential at a much lower dose than that required to

block A-fiber conduction (416), compatible with the critical
Faster
Faster
role of Nav1.7 in C-fiber conduction. It can act as an anal-

UC
NT
NT
NT
NT
NT
NT
gesic in behavioral pain assays but has a narrow therapeutic

Depol
Depol Depol
Depol
Depol
index, which has been recently improved by a new genera-

V1/2
UC
UC
UC
UC
UC
tion of toxins (172). HWTX-IV did show inhibition of

pain-related hypersensitivity in inflammatory and neuro-
V1/2
act
UC
UC
UC
UC
UC
M932L; V991L* 21698661 2794A⬎C; UC
UC
UC
pathic pain models (296). Pn3a showed efficacy in vitro in

Pubmed ID Nucleotide
reducing TTX-S currents in small-diameter DRG neurons

21698661 1867G⬎A
22826602 2215A⬎G
25209274 3836G⬎C
2971G⬎T
21698661 2159T⬎A
29176367 4787C⬎T
29176367 5555T⬎C
22826602 554G⬎A
22136189 684C⬎G
and nociceptive synaptic transmission in spinal slices; how-

ever, it did not have an analgesic effect in acute pain models
using natural stimuli (109).
Gp-Tx1 peptide is another toxin purified from Tarantula

venom, which was also recently shown to potently inhibit
Nav1.7 (327) with promising selectivity and has been used
Amino Acid
as a starting point for modified peptides with improved

potency and selectivity (328). Gp-Tx1 shares the inhibitory
M1852T
R1279P
D623N
T1596I
R185H
I228M
I720K
I739V
cysteine knot motif of ProTx-II and HWTX-IV and binding

maps to the voltage sensor of domain II of Nav1.7 (326)
(similarly to ProTx-II and HWTX-IV).

BENNETT ET AL.
Assays have now been developed for medium throughput 3. Small molecules
screening of toxins; for instance Klint et al. (265) screened
venom from 205 spiders of 13 different taxonomic species. A number of companies are developing small-molecule in-
This fluorescent assay using a human cell line (SH-SY5Y) hibitors to selectively block Nav1.7, as these would have the
revealed that as many as 40% of venoms may contain at great advantage over toxins or antibody therapeutics of oral
least one inhibitor of Nav1.7 (note that a conservative esti- dosing [for a recent review, see Rivara and Zuliani (386)].
mate is that each venom may contain at least 200 peptides). Nav blockers in current clinical use, such as local anesthetics
The authors found seven new members of the NaSpTx- and TTX, interact with the pore region. There is a high level
family 1. One of these peptides, Hd1a, was studied further of homology within the pore (formed by S5 and S6) be-
and was shown to have selectivity for hNav1.7, with the tween different Nav ␣-subunits, making the development of
exception that it also inhibits hNav1.1 with equal potency. selective blockers targeting the pore module challenging.
Like Huwentoxin, Hd1a appears to bind to the S3b-S4 Newer approaches, therefore, are focusing on agents that
helix-turn-helix (the paddle motif). Residues important for target other regions of this channel with less homology,
interaction of the NaSpTx-family1 peptides with Nav1.7 such as the voltage sensor, and this is leading to compounds
were in a structurally well conserved region of the COOH- with greater subtype specificity (309). These arylsulfon-
terminal region and loop. amide inhibitors bind to an extracellular site on the voltage
sensing region of domain IV, and recent structural data
Other toxins that interact with Nav1.7 have been purified demonstrates that this is through binding to the fourth gat-
from centipede and cone snail. The peptide ␮-SLPTX- ing charge residue (R4) within S4 (7). This traps the voltage
Ssm6a (in mature form a peptide of 46 amino acids) was sensor in an activated conformation, which stabilizes a non-
purified from the toxin of the Chinese red-headed centipede conductive state of the channel, probably through preven-
(Scolopendra subspinipes mutilans) (493). This toxin inhib- tion of inactivation. This structural information should
ited Nav1.7 with an IC50 of 25 nM by shifting the voltage help in the rational design of other small molecules target-
dependence of activation to more depolarized potentials. It ing the voltage-sensing domain of Nav1.7. Indeed, other
had no effect on hNav1.3, hNav1.4, hNav1.5, and hNav1.8, novel arylsulfonamides have recently been described: the
and only inhibited hNav1.1, hNav1.2, and hNav1.6 at high clinical compound, PF-05089771, and the structurally re-
concentrations. Furthermore, it was shown to inhibit pain- lated preclinical tool compound, PF-05198007, have been
related behavior in the mouse in terms of reflex withdrawal demonstrated to have high Nav1.7 selectivity by binding to
to noxious heat and the response to intraplantar formalin. the voltage-sensing region in domain IV (12). This study
investigated the role of Nav1.7 in action potential electro-
2. Antibodies genesis using these specific Nav1.7 inhibitors. They provide
pharmacological evidence that, while Nav1.7 acts as a
Given the fact that toxins that bind to the “paddle motif” threshold channel as previously described (466), a reduc-
in domain 2 required for voltage sensing had shown ef- tion in action potential peak height and slope is observed in
ficacy in inhibiting activation of Nav1.7, it was a logical the presence of PF-05198007, suggesting an additional role
step to develop monoclonal antibodies targeting this re- of Nav1.7 in contributing to the upstroke of the action
gion. This motif shows diversity between different ␣-sub- potential (12).
units, enabling the development of the monoclonal anti-
body SVmab1 (278). This showed potent and selective Some agents have been trialed in small case series of patients
inhibition of Nav1.7. Inhibition of Nav1.7 currents was with known mutations in Nav1.7 resulting in IEM (189);
via stabilization of the closed state in a similar manner to however, such studies are currently underpowered. Conver-
ProTx-II and HwTx-4, although, unlike these toxins, in- gence pharmaceuticals have completed phase 2 trials of
hibition occurred in a state-dependent manner (i.e., in- “BIIB074,” their state dependent blocker of Nav1.7; how-
creased at higher frequency of activation). It reduced the ever, the selectivity of this compound for Nav1.7 has been
sodium current in small- (but not large-) diameter DRG questioned (110). A double-blind, placebo-controlled, ran-
cells, as well as action potential generation. In preclinical domized withdrawal phase 2a trial in trigeminal neuralgia
models, this antibody could suppress inflammatory and did not show a significant difference between treatment and
neuropathic pain-related behavior and, interestingly, control on the primary outcome, which was treatment fail-
also itch. This appears, therefore, to be a promising ap- ure; however, secondary end points were met, including a
proach in developing biological agents with a view to significant reduction in number of paroxysms and average
human therapeutics. One caveat is that other groups have daily pain score (504). The second trial is in painful lumbo-
attempted to generate monoclonal antibodies to the same sacral plexopathy (https://clinicaltrials.gov/ unique identifi-
peptide sequence and failed to see any binding of such ers NCT01561027) and has not yet been formally pub-
antibodies to Nav1.7 or any effect on Nav1.7 relative to lished. These studies are relatively small, and larger trials
isotype controls (293). are needed to definitively assess efficacy.

IV. PAIN AND NaV1.8 (321). These data suggest dynamic regulation of Nav1.8
expression in sensory and motor systems during develop-
Nav1.8 is encoded by the gene SCN10A and is preferentially ment of aging.
expressed in peripheral sensory neurons. It has been shown
to shape action potentials in these neurons and contribute Nav1.8 is widely expressed in small-diameter DRG neu-
to pain phenotypes in animal studies and, more recently, in rons, most of which are functionally identified as nocicep-
humans. It has been also implicated in pathologies other tors, but was also reported in a small percentage of
than pain. In this section, we review studies of Nav1.8, medium- and large-diameter neurons (3, 127, 441). Studies
including its pattern of expression, subcellular localization, in DRG neurons in rats and mice have reported Nav1.8 in
and modulation, which have yielded new insights into this 50 – 85% of C fibers, but only 9.5–13% of those with A
channel and its role in action potential firing in neurons. We fibers (2, 14, 338). Electrophysiological evidence for
also discuss the role of Nav1.8 as an important contributor Nav1.8 currents in DRG neurons suggested wider expres-
to painful channelopathies in humans. sion in large-diameter neurons (127, 379), including cuta-
neous afferents that were labeled by fluorogold tracing
(387, 388). Using a Nav1.8-Cre-dependent reporter mouse,
A. Nav1.8 Sequence, Expression Pattern,
Shields et al. (428) showed that Nav1.8 is present in 75% of
and Subcellular Distribution
DRG neurons, with expression in ⬎90% of neurons with
nociceptor marker, in low-threshold C-type mechanorecep-
1. Tissue-specific distribution of Nav1.8
tors, and in a significant population (40%) of myelinated
A-fiber neurons, including low-threshold mechanorecep-
Nav1.8 is encoded by the gene SCN10A and was termed
tors essential for touch sensation. A wider expression of
SNS (sensory neuron specific) (9) or PN3 (peripheral nerve
Nav1.8 in small- and large-diameter neurons in nodose gan-
3) (406) because it was first identified in peripheral sensory
glia was confirmed in another Nav1.8 Cre-dependent re-
neurons of rat DRG and TRG. Nav1.8 transcripts (9, 406)
porter mouse line (180). The distribution of Nav1.8 in DRG
and protein (127) were found in DRG neurons. Nav1.8 was
neurons was recapitulated using a transgenic mouse line in
not detected in nonneuronal tissues, such as heart and skel-
which EGFP expression was driven by a 3.7-kbp Scn10a
etal muscle, or in the CNS, including brain and spinal cord
proximal promoter fragment, although the green fluores-
(9, 10, 338, 406). In other parts of the PNS, Nav1.8 is
cent signal was not detected in nodose ganglia or other
present in nodose ganglia but absent from superior cervical
ganglia of placodal origin, a finding that could suggest the
ganglion (SCG) (9, 273, 400, 406, 415).
presence of additional regulatory elements for the normal
Using reporter mice in which the reporter protein, either expression of this channel in the different neuronal tissues
␤-galactosidase or the fluorescent protein tdTomato, is pro- (301). These data demonstrate a wide expression pattern of
duced in cells in which the Cre recombinase is expressed Nav1.8 within sensory neurons that is not limited to noci-
from the Nav1.8 locus (3, 441), the expression pattern in ceptors.
sensory neurons was confirmed (428), albeit with the ob-
servation of sporadic positive cells in SCG and myenteric Nav1.8 is diffusely localized along the entire length of non-
neurons (180, 441). ␤-Galactosidase activity appeared at myelinated axons of cornea (48) and sciatic nerves (398). It
embryonic stages E13/14 in mice, indicating the early tran- is localized at free nerve endings, where pain signaling is
scription of Cre recombinase at the Nav1.8 locus (441). initiated, in cornea (48) and epidermis (358). The enrich-
Electrophysiological recording in embryonic rat DRG neu- ment of Nav1.8 in nerve endings of somatosensory C fibers
rons, however, did not detect Nav1.8 currents until E17/ was confirmed in a recent study that identified Nav1.8 to be
E18, which peaked by postnatal day 0 (P0) and remained at the primary TTX-R sodium channel responsible for con-
this level until 6 wk, the last time point in the study (381). ducting compound action potential in distal axons in ro-
The difference in the onset of Nav1.8 expression in these dents and monkey (261). A TTX-R sodium current with
two studies could be related to species differences or to the properties attributable to Nav1.8 is activated following
increased sensitivity of the ␤-galactosidase to detect low TTX-S currents in the axons of cultured DRG neurons
levels of Nav1.8 compared with electrophysiological re- (467). The distribution of Nav1.8 within primary afferents
cordings. is consistent with its major contribution to the sodium cur-
rent that underlies the upstroke of action potentials (51,
Nav1.8 levels in sensory neurons have been shown to dimin- 380).
ish with age in mice. Behavioral analysis showed that aged
mice manifest reduced thermal sensitivity, and biochemical Although naive reporter mice did not show expression of
and immunohistochemistry data demonstrated a reduction Nav1.8 outside the peripheral sensory neurons (3, 441),
in Nav1.8 levels in aged mice (474). Recently, it has been there is evidence for the expression of Nav1.8 in the CNS.
reported that Nav1.8 is detectable in aged motor axons, O’Brien et al. (339) reported immunohistochemical and
where it can contribute to abnormalities in excitability PCR data supporting the expression of Nav1.8 in retinal

BENNETT ET AL.
ganglion cells, but did not report the characteristic slow- sion of ␤1, but a reduction of 30% upon coexpression with
inactivating TTX-R current of this channel. This finding is the ␤3-subunit, and no difference in current density with
contrasted with an earlier study that used PCR and in situ coexpression of ␤2- and ␤4-subunits (512). The difference in
hybridization and did not detect this channel in the retina the cell background of the different heterologous expres-
(170). The transgenic line expressing EGFP behind the sion systems could contribute to the differential effects of ␤3
proximal promoter of Scn10a reported green fluorescence on the current density of Nav1.8. The expression of ␤4
in discrete brain regions and included evidence from qRT- caused a significant hyperpolarizing shift in activation
PCR and immunohistochemistry that supported the expres- (⫺16.7 mV) and inactivation (⫺9.3 mV) of Nav1.8 in HEK-
sion of Nav1.8 in the amygdala, brainstem, globus pallidus, 293 cells (512). Recently, the sodium channel ␤4-subunit
lateral and paraventricular hypothalamus, and olfactory tu- was shown to coimmunoprecipitate with Nav1.8 in DRG
bercle; however, immunohistochemistry showed some neurons, and a COOH-terminal peptide of this subunit has
EGFP-positive but Nav1.8-negative neurons, suggesting been shown to enhance the resurgent current that is pro-
transient expression of Nav1.8 in some cells or the cryptic duced by Nav1.8 (447).
expression of the EGFP because of the absence of transcrip-
tion suppression elements that are present at the native Proteomic analysis has identified additional Nav1.8 channel
Scn10a locus but absent from in the 3.7-kbp fragment partners that regulate channel density in the plasma mem-
(301). Electrophysiological recording in hypothalamic neu- brane. Annexin II light chain (p11), which is expressed at
rons with a robust EGFP signal demonstrated a TTX-R high levels in DRG, was found to bind directly to the amino
current that could be blocked pharmacologically (301) us- terminus of Nav1.8, facilitating its translocation to cell sur-
ing the Nav1.8 blocker A-8033467 (244). These data sup- face and, therefore, increasing functional currents (345).
port the presence of functional Nav1.8 channels in the Conditional deletion of p11 in Nav1.8-expressing neurons
plasma membranes of these CNS neurons. caused a reduction in the Nav1.8 current density (174). The
NH2-terminus of Nav1.8 has been shown to interact with
2. Regulation of Nav1.8 expression by trophic the kinesin molecular motor KIF5B, and this interaction
factors may increase the trafficking of the channel to the cell sur-
face, as well as to distal axons in cultured neurons (443).
The regulation of Nav1.8 levels by NGF was confirmed in This interaction is enhanced by inflammation.
vitro (44, 169), and ceramide, a second messenger for NGF,
increases Nav1.8 current density (509). Direct delivery of Surface expression of Nav1.8 has been shown to be regu-
NGF to cut nerve terminals in vivo restored Nav1.8 levels in lated by intrinsic signals that cause retention of this channel
axotomized DRG neurons (117, 279). The response of in the endoplasmic reticulum (ER) (285, 511). Using bio-
Nav1.8 to NGF in DRG neurons is likely to occur predom- chemical and immunocytochemical assays, the RRR se-
inantly in DRG neurons that express the TrkA high-affinity quence motif in the L1 of Nav1.8 was shown to act as an
receptor, primarily associated with peptidergic DRG neu- ER-retention signal and reduce surface expression (511).
rons in adult rodents (108). GDNF regulates expression of The same study also showed that the COOH-terminus of
Nav1.8 in nonpeptidergic DRG neurons (99, 168, 279). the ␤3-subunit binds to the L1 of Nav1.8 and masks this
However, it is not clear what regulates the expression of motif, which causes an increase in the surface expression of
Nav1.8 in cerebellar Purkinje cells (cPC) in pathological reporter proteins in the heterologous expression system and
conditions such as multiple sclerosis (MS) (discussed be- in native DRG neurons. Another study reported that acidic
low). residues in odd-numbered transmembrane segments of
Nav1.8 caused retention in ER via an interaction with cal-
3. Posttranslational regulation of Nav1.8 expression nexin, and that disruption of this interaction reduces the
degradation of the channel by the proteasome and increases
The association of sodium channel ␤-subunits with Nav1.8 surface expression (285).
reflects an isoform-selective interaction. Although the
knockout of ␤2 in mice reduces the current density of the The L2 of Nav1.8 houses a conserved motif for the interac-
TTX-S channels in DRG neurons, the Nav1.8 current den- tion with ankyrin G (AnkG) (179) and has been shown in a
sity is not affected (300). By contrast, the ␤3-subunit in- genetic screen to interact with a multi-PDZ domain protein,
creased the current density of Nav1.8 and hyperpolarized Pdzd2 (304). The localization of Nav1.8 at nodes of Ranvier
the voltage threshold for activation in the heterologous sys- in the protein zero-deficient and Trembler-J mice (which
tem Xenopus oocytes (423), and the expression of this sub- develop demyelinating neuropathies) (112, 320, 461) sug-
unit in the DRG-derived ND7/23 cell line has been suggested the possible interaction of this channel with ankyrin
gested as the reason for a larger recombinant Nav1.8 cur- G (AnkG), which is necessary for the localization of VGSCs
rent in this heterologous expression system (249). The at nodes of Ranvier (375). Using biochemical assays,
current density of Nav1.8 stably transfected into HEK-293 Nav1.8 was reported to bind to AnkG via the conserved
cells was reported to increase 2.3-fold with the coexpres- motif in L2, albeit with a much lower affinity compared

with the binding of AnkG to an activated Nav1.2 reporter 433). Voltage-clamp recordings of Nav1.8, which was first
construct, and the two proteins colocalize at nerve endings cloned from rat DRG, revealed a slow-inactivating inward
in the skin (323). However, this constitutive interaction of sodium current with depolarized activation and fast inacti-
Nav1.8 and AnkG does not appear to be a major contribu- vation (9, 406). These properties are conserved in human
tor to the accumulation of this channel at nodes of Ranvier DRG neurons (123). Earlier investigators also found the
in myelinated sensory neurons (112, 461); thus a cell-type voltage-dependence of fast inactivation of the Nav1.8-like
specific factor might be necessary for allowing the Nav1.8- current was 30 mV more depolarized for TTX-R than
AnkG complex to localize at nodes of Ranvier in dysmyeli- TTX-S currents in small-diameter DRG neurons (138). Re-
nated motor fibers. Pdzd2 was shown to interact with the cordings of human Nav1.8 channels in rodent DRG neu-
L2 of Nav1.8 and increase channel protein at the cell surface rons show that the half-maximal voltage of fast-inactiva-
and current density, and, interestingly, p11 is upregulated tion (V1/2, fast) is approximately ⫺30 mV for Nav1.8, 50 mV
in Pdzd2-deficient mice, suggesting that interaction of the
more depolarized than most TTX-S channels such as
two proteins is important for the homeostatic expression of
Nav1.7, permitting this channel to remain available to acti-
the channel (424).
vate even when neurons have been depolarized to a RMP
that inactivates other sodium channels (233, 234). Thus the
The carboxyl terminus of Nav1.8 carries another conserved
presence of Nav1.8 can present a critical factor in determin-
motif, the calmodulin-binding isoleucine-glutamine (IQ)
motif, through which calmodulin interacts with the channel ing whether a neuron becomes hyperexcitable or hypoex-
protein; blocking this interaction reduces the current den- citable when the RMP is depolarized (199, 400).
sity by 50 – 65% in rat DRG neurons (79). In contrast,
sodium channel clathrin interacting protein 1A (SCLT1A; The kinetics for fast inactivation of Nav1.8 currents are
also termed CAP-1) recycles Nav1.8 from the cell surface, ~10-fold slower than Nav1.7 (76, 234). Fast inactivation of
probably via a clathrin-mediated endocytic pathway Nav1.8 is incomplete, which results in significant persistent
through a direct binding to its carboxyl terminus that leads currents (FIGURE 11, A AND B) (197). Endogenous Nav1.8
to the formation of a multiprotein complex with clathrin currents in native human DRG neurons (FIGURE 11B) pro-
(289). The interaction between SCLT1A and Nav1.8 was duce a persistent current (6.1% of peak current; FIGURE
specific among subtypes of sodium channels, as indicated by 11C) twice as large as that in native rat DRG neurons (FIG-
50% reduction of Nav1.8 current density compared with URE 11A) (3.4% of peak current) (197). The substantial
negligible reduction of TTX-S sodium currents in DRG neu- amount of non-inactivating Nav1.8 channels could contrib-
rons or the skeletal muscle sodium channel isoform Nav1.4 ute to the massive ramp currents, which are also doubled in
in heterologous expression system (289). amplitude in native human DRG neurons (18.6% of peak
current) compared with rat DRG neurons (9.8% of peak
Ubiquitin protein ligase neural precursor cell expressed, de- current) (FIGURE 11D) (197).
velopmentally downregulated 4 –2 (Nedd4 –2) regulates
channel density at the plasma membrane. Nedd4 –2 binds Nav1.8 undergoes slow inactivation when a depolarization
to Nav1.8 carboxyl terminus via the PXY motif and effec- stimulus is applied for a sustained period of 10 s (50, 342,
tively abolishes sodium currents when coexpressed in Xe- 397). The entry of Nav1.8 into slow inactivation was faster
nopus oocytes, but fails to do so when the Tyr residue of and recovery from it was slower in IB4⫹ neurons compared
PPSY motif was mutated to an Ala residue (Y1921A) (173). with IB4⫺ neurons (80). Cooling (from 30°C to 10°C)
Interestingly, the Nedd4 isoform is less effective than the markedly hyperpolarizes the slow inactivation of Nav1.7
Nedd4 –2 in inhibiting the Nav1.8 current. Downregulation and TTX-S current in DRG neurons without affecting that
of Nedd4 –2 and increase of Nav1.8 currents were shown in
of Nav1.8 or TTX-R current, which accounts for mainte-
rat DRG in the SNI model associated with aggravating neu-
nance of Nav1.8 availability during the cold (517). The cold
ropathic pain, and the restoration of Nedd4 –2 expression
resistance of slow inactivation of Nav1.8 endows it a special
using virus infection reduced Nav1.8 currents and alleviated
role in nociceptors for the perception of noxious cold and
SNI-induced mechanical allodynia (60, 274).
mechanical stimulation at low temperatures (517).
B. Biophysical Properties of Nav1.8 Expression of human and rat Nav1.8 in DRG neurons from
Nav1.8-null mice also showed that human Nav1.8 currents
1. TTX-resistance, depolarized activation, and exhibit hyperpolarized activation and depolarized fast inac-
inactivation tivation compared with rat Nav1.8 currents, creating a
larger window for human Nav1.8 (197). In addition, slower
The aromatic residue (Phe/Tyr) in the S5– 6 extracellular kinetics for open-state inactivation enable the human
linker in domain 1 critical for high-affinity binding of TTX Nav1.8 channel to remain open longer (197). These obser-
(410) to sodium channels is substituted by a Ser residue in vations suggest a greater effect of human Nav1.8 on neuro-
Nav1.8, which confers TTX resistance on Nav1.8 (9, 406, nal activity than predicted from rodent studies.

BENNETT ET AL.
FIGURE 11. Human dorsal root ganglia (DRG) neurons

demonstrate larger Nav1.8 persistent currents. A: repre-
sentative traces of Nav1.8 currents from native rat DRG
neurons. B: representative traces of Nav1.8 currents from
native human DRG neurons. C: Nav1.8 currents in human
DRG neurons produce larger persistent currents than rat.
D: Nav1.8 currents in human DRG neurons produce larger
ramp currents than rat. [Adapted with permission from
Han et al. (197).]
A recent study by Zhang et al. (505) also reported biophys- TTX-R current in rat DRG neurons (25% of peak current)
ical properties of TTX-R currents in human and rat DRG reported in the Zhang et al. study, which has not been
neurons. Consistent with the earlier results of Han et al. previously reported in any other study (9, 10, 99, 100, 104,
(197), steady-state activation of the TTX-R currents was 185, 187, 197, 338, 406). Despite these differences, the
hyperpolarized by ~5 mV in human DRG neurons com- Han et al. (197) and Zhang et al. (505) studies are impor-
pared with rat DRG neurons. Steady-state fast inactivation, tant for beginning to establish a baseline for future studies
however, was reported to be hyperpolarized in human DRG of human Nav1.8 in its native environment.
neurons compared with rat. Moreover, Zhang et al. re-
ported a much larger persistent current in rat DRG neurons, 2. Repriming and resurgent currents
which reached 25% of the peak current, and a significantly
smaller TTX-R persistent current in human DRG neurons.
Nav1.8 inactivates from the open-state with slow kinetics,
The discrepancies between the Han et al. (197) and Zhang
and it recovers from inactivation rapidly (9, 10). These
et al. (505) results may reflect different methodologies that
findings are in agreement with early observations in rat
were implemented for recording of the sodium currents.
DRG neurons in which the Nav1.8-like TTX-R current in-
The study by Han et al. recorded the TTX-R current in
activation is more than 10 times slower than that of the
human and rat DRG neurons with 500 nM TTX in the bath
TTX-S current, while repriming is more than 10 times faster
and by holding the neurons at – 60 mV to inactivate the
(138). For example, at a voltage typical of neuronal RMP
persistent Nav1.9 currents that are native to these neurons.
(⫺67 mV), the time constants for repriming kinetics for
By contrast, Zhang et al. did not include TTX in the bath
TTX-R and TTX-S sodium currents was 3.8 ms and 51 ms,
solution and held the neurons at ⫺80 mV. Although Zhang
respectively (138). The tetrapeptide SLEN in the S3-S4
et al. reported that steady-state availability curves were de-
linker of D4 was reported as a molecular determinant for
termined for every neuron, which would have provided an
rapid repriming of Nav1.8 because inserting the SLEN res-
estimation of the currents that remain available at – 80 mV,
idues into the corresponding position of Nav1.4 caused the
the TTX-R currents reported in this study may include both
chimera channel to reprime twice as fast as the WT channel
Nav1.8 and Nav1.9 currents. It is also possible that contam-
(120). A slow TTX-R resurgent current in rat DRG neurons
ination by residual TTX-S currents, especially in neurons
is produced by Nav1.8 and was sensitive to the Nav1.8
with a large TTX-S conductance, and Nav1.9, which has a
specific blocker A-803467 (447).
more hyperpolarized steady-state inactivation compared
with Nav1.8, could have contributed to the hyperpolarized
steady-state inactivation that was reported for human 3. Contribution of Nav1.8 to action potentials
Nav1.8 by Zhang et al. Another source of divergence in the
data could come from the larger number of donors of the Nav1.8 contributes the majority (58 –90%) of the inward
human DRG tissue that was included in the Zhang et al. current during the rising phase of an all-or-none action
study, compared with that in the Han et al. study. However, potential in nociceptive sensory neurons (51, 380). Nav1.8
what is not easily explicable is the very large persistent fast inactivation is depolarized and thus does not inactivate

completely during the falling phase of action potential,

thereby contributing to the action potential duration more
than other TTX-S sodium channels. Thus Nav1.8 contrib-
utes substantially to an inflection (hump) in the falling
phase (51, 81). Nav1.8 contributes most of the current in
subsequent spikes, probably due to the depolarized inacti-
vation and rapid repriming of this channel (50). Because of
its voltage dependence, TTX-R resurgent sodium currents
might also contribute to action potential broadening (447).
The fast recovery of Nav1.8 from fast inactivation and the
Nav1.8 resurgent currents are implicated in the subthresh-
old oscillation of RMP and repetitive firing in DRG neurons
(81, 447).
Current-clamp recordings in human DRG neurons or

Nav1.8-null mouse DRG neurons expressing human
Nav1.8 channels showed significantly prolonged and
unique firing patterns of action potentials (197). Roughly
one-fourth (23%) of DRG neurons expressing human
Nav1.8 channel fired prolonged action potentials with a
half-width longer than 10 ms, whereas none of the native
rat DRG neurons or Nav1.8-null mouse DRG neurons ex-
pressing rat Nav1.8 channels did. Human Nav1.8 increased
the average action potential half-width by 69% compared FIGURE 12. Distinct properties of human Nav1.8 channel. A:
with rat Nav1.8. Moreover, bursting events were recorded responses of representative dorsal root ganglia (DRG) neurons ex-
pressing rat (top) and human (bottom) Nav1.8 channels to 500 ms
only in neurons expressing human Nav1.8. The expression of 200-pA depolarization current injection. B: comparison of re-
of human Nav1.8 produced a threefold increase in the pop- sponses (no. of impulses evoked by a 500-ms stimulus) in DRG
ulation of spontaneously firing cells, where burst firing was neurons expressing human or rat Nav1.8 channels across a range
also captured. In contrast, spontaneously-firing DRG neu- of step current injections from 50 to 500 pA. *P ⬍ 0.05. C:
rons expressing rat Nav1.8 channels did not demonstrate comparison of action potential (AP) half-width between human
Nav1.8 group and rat Nav1.8 group. ***P ⬍ 0.001. AP width in
burst-like responses. Given the same external stimuli, DRG each neuron was measured after applying a kinetic model of human
neurons expressing human Nav1.8 fired significantly more Nav1.8 and subsequently in the same cell after applying a kinetic
action potentials than those expressing rat Nav1.8 (FIGURE model of rat Nav1.8 (symbols connected for each neuron). Two
12, A AND B). larger symbols indicate means ⫾ SE. D: comparison of the half-
width of AP between rat and human DRG neurons. Two larger
symbols indicate means ⫾ SE. ***P ⬍ 0.001. [Adapted with per-
Using dynamic-clamp recording, a single neuron was en- mission from Han et al. (197).]
dowed with Nav1.8 conductance from either human or rat
subtype. In this analysis, the broadening of action potential
by human Nav1.8 was confirmed (FIGURE 12C), with re- tion of the channel (80, 458). Slow inactivation of Nav1.8 is
cordings from native human DRG neurons demonstrating a enhanced in nonpeptidergic (IB4⫹) nociceptors compared
broad action potential (FIGURE 12D). The unique biophys- with peptidergic (IB4⫺) nociceptors (80). The faster entry of
ical properties of human Nav1.8 produced spontaneous fir- Nav1.8 into slow inactivation and its slower recovery from
ing in 23% of DRG neurons compared with 0% when rat it in IB4⫹ neurons compared with IB4⫺ neurons is consis-
Nav1.8 conductance was injected into the cell. The rheo- tent with the observation that IB4⫹ neurons display stron-
base for evoked firing was reduced by almost 50% after ger adaptation in response to sustained stimulus (80).
injecting human Nav1.8 conductance compared with rat
Nav1.8. The distinct properties of human Nav1.8 and the
distinct action potential characteristics in human DRG neu- C. Role of Nav1.8 in Pain
rons should, therefore, be taken into account when extrap-
olating from rodent studies of pain to humans. The critical role of Nav1.8 in repetitive firing, and its local-
ization in free nerve endings, where the response to external
Adaptation of action potentials (i.e., progressively reduced stimuli is integrated and action potentials are initiated, sug-
overshoot in a train of action potentials) during repetitive gests that Nav1.8 can play an important role in nociception
firing in a subpopulation of DRG neurons occurs with a and chronic pain. Animal and human studies have sup-
time course similar to that of development of slow inacti- ported a role for Nav1.8 in several pain modalities, and
vation of Nav1.8 (50) and increased use-dependent inhibi- targeting Nav1.8 has ameliorated pain symptoms in animal

BENNETT ET AL.
models. The role of Nav1.8 in nociception and chronic pain of Nav1.8 in pain related to damaged axons was strongly
conditions will be discussed in detail below. supported by the reduction in the proportion of spontane-
ously active fibers by ⬎50% in neuromas formed by section
1. Role for Nav1.8 in nociception of the saphenous nerve in Nav1.8-null mice compared with
Natural selection has provided compelling evidence for the their WT littermates (395). Nerve injury, for example spinal
role of Nav1.8 in nociception. Grasshopper mice (Onych- nerve ligation, results in the activation of p38 mitogen-
omys torridus), which are native to the Arizona dessert, are activated protein kinase (MAPK) (414), and inhibiting p38
less sensitive to the venom of the bark scorpion (Centru- MAPK reversed the CCI-induced heat hyperalgesia (341).
roides sculpturatus) on which they feed, than the more sen- Activated p38 has been shown to increase Nav1.8 current
sitive laboratory mice (Mus musculus) (392). Functional density in DRG neurons (35, 237). The observation by
assays determined that the Nav1.8 channels from O. torri- Black et al. (45) that Nav1.8 channels and activated p38
dus mice are inhibited by the venom of the bark scorpion, accumulate within the nerve terminals of painful human
whereas these channels from M. musculus are not, and that neuromas suggests that the colocalization of Nav1.8 and
application of the venom to DRG neurons in culture inactivated p38 MAPK at the neuroma contribute to sponta-
creases firing of M. musculus neurons but inhibits firing of neous pain, brush-evoked allodynia and pinprick hyperal-
the O. torridus neurons, and injection of the venom signif- gesia, and reduced thresholds for thermal stimuli that is
icantly reduces the characteristic response to the injection of detected at these sites (333).
0.5% formalin into the hindpaw of O. torridus (393). This
experiment of nature shows that inhibition of Nav1.8 can Levels of Nav1.8 were found to be reduced in both large and
lead to analgesia. small DRG neurons after axonal injury (107, 116, 344,
434). In contrast to reduced Nav1.8 expression and TTX-R
Hypermorphic mutation of an allele of SCN10A, the gene currents in injured afferents after spinal nerve ligation, a
that encodes Nav1.8, induces a dominant neurobehavioral striking increase in Nav1.8 expression and TTX-R currents
phenotype termed Possum, characterized by tonic immobil- was observed in uninjured neurons, suggesting that Nav1.8
ity triggered by pinching the skin at the back of the neck was redistributed to the axons of uninjured unmyelinated
(“scruffing”), a behavior likened to the temporary arrest of afferents (186, 507). The redistribution of Nav1.8 follow-
whole body movement occurring when prey animals come ing peripheral axotomy has also been reported in humans
under attack by a predator. Compared with WT littermates, (90). In brachial plexus injury patients, Nav1.8 protein was
the peak current density of Nav1.8-like TTX-R sodium cur- reduced in sensory cell bodies of cervical DRG whose axons
rents is significantly increased, accompanied by decelera- were avulsed from spinal cord but increased in peripheral
tion of activation and open-state inactivation in Possum fibers proximal to the site of injury (90). The resultant in-
mice (53). Using the skin-nerve preparation from the Pos- crease in TTX-R sodium currents was consistent with ab-
sum mouse, mechanically evoked action potential firing errant hyperactivity in uninjured C fibers (507) and atten-
was shown to be increased in subclasses of A␤, A␦, and C uation using Nav1.8 antisense ODNs (186, 250, 275, 492),
fibers (178). natural toxin (␮O-conotoxin MrVIB) (137), and synthetic
compound (A-803467) (244) with selectivity for Nav1.8
By contrast, loss-of-function of Nav1.8 attenuates pain be- over other VGSCs alleviated neuropathic pain in rodent
havior. Nav1.8-null mice manifest attenuated response to models.
cold and mechanical stimuli, and thermal hyperalgesia in-
duced by intraplantar injection of carrageenan developed The reduction in the Nav1.8 in DRG neurons (104, 116,
more slowly, compared with WT littermates (10). Nav1.8- 434) was specific to peripheral axotomy, since the total
null mice also showed blunted pain-related behavior to in- TTX-R current in centrally axotomized neurons remained
tracolonic capsaicin and mustard oil, which provoked tis- unaffected (434) and is in contrast to the accumulation of
sue damage and sensitized nociceptors in WT mice (276), Nav1.8 in the injured tips of axons at neuromas in patients,
consistent with the expression of Nav1.8 in DRG neurons as discussed above. However, other studies using a modified
that innervate the colon (187). Hyperexcitability in DRG nerve transection surgery did not show the reduction of
neurons was absent in Nav1.8-null mice with jejunitis in- Nav1.8 currents (1). The main difference between these le-
duced by infection with Nippostrongylus brasiliensis (Nb) sions is that studies that reported axotomy-induced reduc-
(216). Antisense ODN targeting Nav1.8 or selective phar- tion in Nav1.8 isolated the cut end of the sciatic nerve from
macological blockade of Nav1.8 has efficiently attenuated the surrounding tissue by enclosing the proximal end in an
mechanical allodynia and thermal hyperalgesia following inert silicon cuff, thus preventing target-derived trophic
intraplantar CFA injection in animal model (244, 250). support, and causes a downregulation of Nav1.8 mRNA,
2. Nav1.8 and neuropathic pain protein, and current in injured neurons (104, 116, 434),
whereas the Abdulla et al. study (1) used a surgical method
Nav1.8 channels have been shown to accumulate within in which transected nerves were not prevented from contact
nerve endings in neuromas (90, 271, 498). The critical role with surrounding tissue. As discussed earlier, supplying the

cut nerves with trophic factors can restore the expression of 418). These inflammatory mediators activate downstream
Nav1.8 currents (117, 279). Thus the divergent findings in signaling cascades, including protein kinases that can act on
some animal models and the human data can be explained effector molecules that include Nav1.8 (35, 142, 184, 187,
by methodological differences in the surgical method used 248, 404, 485, 491, 514). Multiple inflammatory media-
to produce injury. tors, including prostaglandin E2 (PGE2), adenosine, sero-
tonin, tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-
Diabetic neuropathy is a common form of peripheral neu- 1␤), CXCL12 and CXCL13, and endothelin-1 (ET-1) have
ropathy, with many patients going on to develop neuro- been shown to induce phosphorylation of Nav1.8, modu-
pathic pain (159). In the STZ-induced diabetic rat model, lating current density and gating properties and altering the
which manifests mechanical allodynia and thermal hyper- firing properties of DRG neurons.
algesia, Nav1.8 expression was noticeably reduced (229,
344). However, TTX-R sodium currents from small-diam- Acute administration of PGE2, adenosine, or serotonin in-
eter DRG neurons were increased, accompanied by hyper- creases the amplitude of Nav1.8-like TTX-R sodium cur-
polarized shifts in voltage dependence of activation and rent, hyperpolarizing its voltage dependence of activation,
steady-state fast inactivation, presumably due to elevated and accelerates its rate of activation and inactivation in
serine/threonine phosphorylation of Nav1.8 in the diabetic DRG neurons (185, 187). Alteration in the biophysical
condition (229). Methylglyoxal, a glucose metabolite that properties of Nav1.8-like TTX-R sodium current by PGE2,
accumulates in diabetes, can also modify Nav1.8 by depo- which involves activation of PKA and PKC, reduces rheo-
larizing its voltage dependence of fast inactivation by ~10 base for firing a single action potential and increases firing
mV, an effect that increases channel availability (34). Short- frequency of action potentials to a suprathreshold stimulus,
term (3-h) exposure of DRG neurons to methylglyoxal re- leading to hyperexcitability in rat DRG neurons (142, 184).
sulted in depolarization of RMP by ~8 mV, concomitant PKA and PKC are known to phosphorylate Nav1.8 at spe-
with almost 50% reduction in rheobase, all of which con- cific serine residues within the L1 of the channel (167, 469).
tributes to hyperexcitability in DRG neurons that underlies
the mechanism of metabolically driven hyperalgesia (34). TNF-␣, an important proinflammatory cytokine that serves
Blocking of Nav1.8 in diabetic rats with A-803467 admin- as a trigger for activation of other cytokines and growth
istered intraperitoneally or by intraplantar injection has
factors in response to inflammation, acutely enhances
been reported to attenuate mechanical allodynia and ther-
Nav1.8-like TTX-R currents in DRG neurons by interacting
mal hyperalgesia, and this effect was more effective than
directly on TNF receptor 1 through activation of p38
treatment with lidocaine (312).
MAPK (248). Similarly, another proinflammatory cyto-
kine, IL-1␤, was reported to increase the peak amplitude of
3. Nav1.8 and inflammatory pain
TTX-R sodium current in small DRG neurons and to accel-
Multiple studies have supported the involvement of Nav1.8 erate activation kinetics in a p38 MAPK-dependent manner
in the development of inflammatory pain. Following injec- (35). Although the voltage dependence of either activation
tion of carrageenan into the hindpaw of the rat, Nav1.8 or fast inactivation for Nav1.8-like TTX-R current was not
transcripts almost doubled and TTX-R current density sig- altered, IL-1␤ depolarized its slow inactivation and en-
nificantly increased in small DRG neurons projecting to the hanced persistent current, which increases nociceptive neu-
inflamed limb (449). In another inflamed rat model where ronal excitability and contributes to peripheral sensitiza-
peripheral inflammation was induced by intraplanter injection (35). Levels of the chemokine CXCL13 are also in-
tion of CFA, Nav1.8 immunoreactivity increased by 1.5- creased in DRG neurons after CFA-induced inflammation,
fold in unmyelinated and 6-fold in myelinated axons in leading to increased activation of p38 MAPK and the in-
digital nerves of inflamed rat hindpaws (85). CFA-induced crease in the Nav1.8 current density (485). The p38-medi-
inflammation has been shown to also act via activation of ated increase in Nav1.8 current density, which was not ac-
protein kinase B/Akt, which increases levels of the Nav1.8 companied by changes in voltage dependence of activation
channel protein in DRG neurons (286). The mechanisms or fast inactivation, is caused by phosphorylation of two
that underlie this increase in Nav1.8 expression levels are p38 phospho-acceptor serine residues in the L1 loop of the
not well understood. Nav1.8-like TTX-R currents in noci- Nav1.8 channel (S551 and S556), and the substitution of
ceptive DRG neurons also increased significantly in rodent these residues by alanine blocks the increase in the Nav1.8
models of visceral inflammation, such as mild gastritis (32, current density (237).
33), urinary tract inflammation (499), and colitis (30).
Nav1.8 channels are also regulated by activated Erk1/2
The findings described above support the conclusion that MAPK (16, 491). Nav1.8 current density is increased after
inflammation-induced signaling cascades alter Nav1.8 ex- treatment of DRG neurons with basic fibroblast growth
pression levels. The local release of inflammatory mediators factor, a cytokine that is involved in wound healing, via
by immune and injured cells induces sensitization of DRG activation of Erk1/2 MAPK (16). A study using bee venom
neurons, which contributes to the pain phenotype (236, intraplantar injection to induce persistent inflammatory re-

BENNETT ET AL.
sponse identified the chemokine CXCL12 (also called stro- Nav1.8 mutation in DRG neurons caused a reduction in
mal cell-derived factor 1) and its receptor CXCR4 as play- rheobase and increased in firing frequency of action poten-
ing an important role in the ensuing persistent pain, which tials in response to suprathreshold stimuli. L554P also tri-
appears to be mediated by enhancing Nav1.8 currents in an pled the proportion of spontaneously firing neurons. The
Erk1/2-dependent manner (491). second mutation, A1304T, substitutes a highly conserved
residue in DIII S5. This mutation hyperpolarized activation
ET-1 has been implicated in the pathogenesis of tissue in- by 6 mV and shifted the peak of the ramp response to a
flammation and nociception. ET-1 contributes to carra- similar degree. Expression of the A1304T mutant in DRG
geenan or E. coli lipopolysaccharide-induced inflammatory neurons depolarized resting potential by 5 mV, reduced
pain in the knee joint in rats (106). The effects of ET-1 are rheobase, and increased firing frequency.
mediated by ETA and ETB receptors. ETB receptor knock-
out (⫺/⫺) mice did not manifest pain symptoms induced by Since that initial report, two additional gain-of-function
phenylbenzoquinone and experienced much reduced cuta- SCN10A variants identified in SFN patients were later func-
neous inflammatory response to topical arachidonic acid tionally profiled: G1662S in the P-loop within the extracel-
application (192). Acute exposure of rat DRG neurons to lular linker between transmembrane segments 5 and 6 of
ET-1 results in gain-of-function changes in gating of domain IV (201) significantly impaired fast inactivation
Nav1.8-like TTX-R currents predominated by a marked (FIGURE 14A), thereby enlarging the window current (FIG-
hyperpolarizing shift in the voltage dependence of activa- URE 14B), enhanced recovery from inactivation near RMP,
tion (514). and increased the population of neurons that fired sponta-
neously by 3.5-fold (201). The I1706V mutation, in trans-
D. Nav1.8 and Inherited Human Pain membrane segment 6 of domain IV, hyperpolarized activa-
Disorders tion, reduced rheobase (FIGURE 15A), and increased the
population of repetitively firing neurons (FIGURE 15B) and
Accumulating evidence has linked mutations Nav1.8 to action potential firing frequency in response to external
SFN (FIGURE 13 and TABLE 5). A pioneering study investi- stimuli (FIGURE 15C) (234). Hyperexcitability in DRG neu-
gating the connection between Nav1.8 and SFN was per- rons resulting from expressing Nav1.8 gain-of-function
formed in 104 patients with idiopathic painful predomi- variants (TABLE 5) provides a pathological basis for the pain
nantly SFN, all negative for mutations in SCN9A (151). experienced by patients carrying these mutations.
Genomic screening identified seven missense variants in
SCN10A in nine patients. Three mutations fulfilled selec- Several other variants in Nav1.8 from patients with SFN
tion criteria for functional analysis and were subsequently have been reported (151), but their contribution to the pa-
characterized in DRG neurons by whole cell voltage-clamp thology is not clear, because they have not been functionally
and current-clamp recording. The L554P mutation in L1 tested. A mutation in Nav1.8, M650K in L1, has been re-
hyperpolarized activation, accelerated recovery from fast ported from a patient with EM (259). This study reported
inactivation at membrane potentials close to neuronal voltage-clamp recording that showed a hyperpolarizing
RMP, and enhanced slow-ramp currents. Expression of this shift in steady-state fast inactivation, and current-clamp
FIGURE 13. Location of pain-associated SCN10A mutations. Nav1.8 ␣-subunit structure shows the location
of amino acids associated with human pain disorders.

Suprathreshold
Response to
Increased
Increased
Increased
Injection
Current
Reduced
NT
RMP Threshold
Reduced
Reduced
Reduced
Current
UC
NT
Depol
Depol
FIGURE 14. G1662S Nav1.8 mutation impairs fast inactivation
UC
UC
UC
and produces hyperexcitability in dorsal root ganglia neurons. A:
comparison of steady-state fast inactivation for wild-type (WT) and
Increased
Increased G1662S channels. G1662S mutation impairs steady-state fast in-

Activity
activation by 6.8 mV, which is the difference between the V1/2

inactivation of WT and mutant channel. B: comparison of the overlap
UC
UC
NT
of activation and fast-inactivation curves between WT and G1662S.

TABLE 5. Electrophysiological characteristics of published SCN10A mutations
[Adapted with permission from Han et al. (201).]

Decreased
Current
recordings that showed broader action potential width and

Increased UC
UC
UC
Increased UC
reduced evoked firing. It is not clear how these loss-of-

Hyperpol
Hyperpol
Ramp
function attributes of the mutant Nav1.8 could contribute

to the pain symptoms in this patient, and the authors sug-
NT
gest that it is possible that this mutation in Nav1.8 is in fact

Repriming Inactivation Deactivation Resurgent
playing a protective effect, and that the patient would have

NT
NT
NT
NT
NT
suffered more severe pain in the absence of this mutation

(259).
Study of a more common variant in Nav1.8 has provided

UC
NT
NT
NT
NT
evidence supporting the contribution of this channel to pain

in normal subjects. Duan et al. (131) tested the hypothesis
that rs6795970 (G⬎A; p.Ala1073Val) affects human pain
sensitivity, and they demonstrated an association between
Slow
rs6795970 and lower mechanical pain sensitivity in a dis-

UC
UC
UC
NT
NT
covery cohort (496 subjects) and confirmed it in a larger

replication cohort (1,005 female subjects). The minor allele
shifts channel activation by ⫺4.3 mV, a proexcitatory at-
Faster
Faster
tribute, and accelerates inactivation and decreases persis-

UC
UC
NT
tent currents, both anti-excitatory attributes. Expression of

the minor allele in DRG neurons reduced repetitive firing,
Hyperpol
inact
consistent with higher thresholds for mechanical pain that

V1/2
Depol
were found in carriers of this SNP. Taken together, these

UC
Hyperpol UC
Hyperpol UC
studies in humans validate Nav1.8 as a potential therapeutic

Pubmed ID Nucleotide V1/2 act
target for development of new therapies for pain.

UC
UC
UC
A1304T 23115331 3910G⬎A
G1662S 24006052 4984G⬎A
23986244 5116A⬎G
E. Nav1.8 in Pathologies Other Than Pain

27598514 1949T⬎A
23115331 1661T⬎C
Nav1.8 has been shown to be expressed in cPC under

inactivation, respectively.
pathological conditions. Using in situ hybridization and

immunohistochemical assays, Nav1.8 was first reported
in the cPC in the taiep rat (a rodent model of CNS demy-
elination), but only after the loss of myelin (41). Nav1.8
mRNA and protein was then reported in cPC in experi-
M650K
I1706V
Amino
L554P
Acid
mental autoimmune encephalomyelitis (EAE) animal

models of MS (39) and in humans with MS. The expres-
sion of Nav1.8 in cultured Purkinje neurons in vitro or in

BENNETT ET AL.
FIGURE 15. I1706V Nav1.8 mutation renders hyperex-

citability in dorsal root ganglia (DRG) neurons. A, left: re-
sponses of a current-clamped DRG neuron transfected with
Nav1.8-wild-type (WT) DNA to a series of subthreshold and
suprathreshold depolarizing current steps. Starting at a
subthreshold stimulus intensity, the current amplitude was
increased in 5-pA increments to an intensity well beyond
threshold. The resting membrane potential (RMP) for this
cell was ⫺55 mV, and the current threshold was 155 pA.
Middle: the same threshold protocol was applied to a DRG
neuron transfected with the I1706V mutant DNA. The
RMP for this cell was ⫺47 mV, and the current threshold
was 35 pA. Arrows with numbers indicate the current step
amplitude used to elicit the labeled response. Right: current
threshold was significantly reduced after expressing
I1706V channels (*P ⬍ 0.001). B: representative record-
ings of repetitively firing action potentials (APs) evoked by
500 pA of injected current for 500 ms in DRG-expressing
I1706V channels. Inset: bar graph shows the proportion of
neurons expressing WT (black) and I1706V (orange) chan-
nels that fire multiple APs in response to the maximally
injected current (500 pA). Numbers to the right of the bar
graph show values for WT and I1706V. *P ⬍ 0.05. C:
summary of firing frequency data. The total no. of APs
elicited by the indicated depolarizing current steps from 25
to 500 pA in 25-pA increments was compared between
two groups of neurons expressing WT or I1706V channels.
*P ⬍ 0.05. [Adapted with permission from Huang et al.
(234).]
mouse models in vivo causes an increase in action poten- ropathy (112). Although analysis of the compound action
tial firing and desynchronizing of the normally stereotyp- potential in this mouse did not reveal the expected resis-
ical firing pattern of these neurons (382, 403, 430). Def- tance to low doses of TTX (112), functional assessment of
icits in coordinated motor function were attenuated fol- conduction in a double knockout mouse carrying null al-
lowing targeting of this channel using a Nav1.8-selective leles of protein zero and Nav1.8 or the pharmacological
blocker (429). The link between Nav1.8 and MS has been block of Nav1.8 in mice heterozygously deficient for protein
strengthened by the recent report of variants in Nav1.8 in high zero led to enhanced conduction and excitability. This ar-
linkage disequilibrium in patients with MS, with carriers of the gues that the ectopic expression of Nav1.8 at nodes is det-
SNP rs6795970; A) demonstrating significantly diminished rimental and has been interpreted as suggesting that block
functional connectivity between the thalamus and midbrain of this channel in CMT patients might be beneficial (320,
and worse performance than carriers of the other allele 390). The mechanism underlying the impaired conduction
(rs6795970, G) in the Multiple Sclerosis Functional Compos- in dysmyelinated fibers carrying Nav1.8 at their nodes is still
ite test consistent with cerebellar dysfunction (389). The unclear.
Nav1.8-mediated altered stereotypic firing pattern of cPC dis-
rupts coordinated motor behavior, while knocking out Nav1.8 Another recent study showed that the SCN10A gene is
attenuated early symptoms in mice with EAE (430). These downregulated by transcription factor 4 in models associ-
data demonstrate a role for Nav1.8 in CNS neurons in animal ated with schizophrenia and Pitt-Hopkins syndrome
models of MS and in humans with MS; this cryptic expression (PTHS) (374). Abnormalities of action potential amplitude
of Nav1.8 contributes to cerebellar deficits in humans and firing frequency were reported in prefrontal cortex
with MS. (PFC) neurons from a Tcf4⫹/tr mouse model of PTHS in
which a significant enhancement of Nav1.8 expression was
Prior to the description of Nav1.8 expression in motoneu- detected. Application of the Nav1.8 blocker A-803467 re-
rons in aged mice (321), dysmyelinating disorders were restored the intrinsic excitability of these PFC neurons.
ported to alter Nav1.8 expression in motoneurons (112,
320, 461). Mice that are deficient in protein zero, which 1. Nav1.8 in heart
cause a severe early-onset dysmyelinating neuropathy anal-
ogous to the Charcot-Marie-Tooth (CMT) neuropathy in Despite initial reports of absence of Nav1.8 in cardiac myo-
humans, display an abnormal localization of this channel at cytes (3, 9, 10, 301, 338, 406, 441), a series of recent
their nodes of Ranvier (320, 390, 461). A similar observa- genomewide association studies (GWAS) suggests a link
tion was made in motor fibers in the Trembler-J mouse, a between myocardial conduction and SCN10A (31, 69, 228,
genetic model of a dominantly inherited demyelinating neu- 362, 439, 463, 494). These observations are supported by

the finding that Possum mice, which carry a gain-of-func- reported to show the presence of Nav1.8 protein in human
tion mutation in Nav1.8, show abnormal cardiac conduc- atrial myocyte, where it distributes in a similar pattern to
tion, including severe sinus bradycardia and irregular R-R connexins, the specialized gap junction proteins of myocar-
intervals (53), as well as prolonged PR interval, consistent dial intercalated disks (152). Yang et al. (494) reported that
with PR interval shortening in Nav1.8-null mice (69). ventricular myocytes exhibited significantly shorter action
GWAS studies also link SCN10A/Nav1.8 to cardiac func- potential duration after A-803467 treatment, most likely
tion and correlate ECG parameters such as PR interval and due to a reduction in late sodium current (i.e., persistent
QRS duration with SCN10A, suggesting a role of Nav1.8 current). Further studies showed antiarrhythmic effects of
variants in atrial and atrioventricular conduction, as well as A-803467 in mouse and rabbit ventricular myocytes.
in susceptibility to atrial fibrillation (AF) and Brugada syn- A-803467 consistently suppressed early afterdepolarization
drome (177, 230). For example, SCN10A variants have in ventricular myocytes, which was triggered by 1-Hz stim-
been identified in 4 –7% of patients with early onset AF ulation under conditions of low extracellular potassium
(242, 411). Although there is no consensus about how and exposure of a sodium channel opener ATX-II (494). It
Nav1.8 contributes to cardiac function or dysfunction, the is possible that modest expression of Nav1.8 channels is
data published to date are consistent with three potential sufficient to play a functionally significant role in cardiomy-
mechanisms for a regulation of the cardiac function by ocyte electrophysiology and cardiac conduction. However,
Nav1.8, as discussed below. this does not explain the absence of reporter proteins in
cardiac myocytes where the Cre recombinase, which is nec-
2. Neuronal hypothesis: expression of Nav1.8 in essary to release the reporter protein from translational sup-
intracardiac neurons pression, is expressed at the Nav1.8 locus (441).
Nav1.8 expression has been detected in murine (468) and

4. Genomic coregulation hypothesis: Nav1.8
canine (74) cardiac ganglionated plexus. The intracardiac
modulates expression of Nav1.5 in cardiomyocytes
ganglia are interconnected clusters of neurons (i.e., gangli-
onated plexuses) located in the interatrial septum and
throughout the atrial epicardium (383). Innervated by the SCN5A encodes Nav1.5, the predominant VGSC in cardi-
vagus nerve, intracardiac neurons project on discrete re- omyocytes, and maps to the short arm of chromosome 3 in
gions of the heart, and their discharge pattern regulates a cluster that also includes SCN10A (68, 124). High-reso-
chronotropic, dromotropic, and inotropic elements of car- lution chromatin conformation capture has been reported
diac function (383). Voltage-clamp studies in the mouse to show the intricate association of the SCN10A intronic
demonstrated that the Nav1.8 selective blocker A-803467 enhancer with the promoters of the SCN5A and SCN10A
(244) reduces Nav1.8 current density in intracardiac neu- and the downstream SCN5A enhancer (463). Compared
rons by ~20%, and hyperpolarizes steady-state fast inacti- with the high-level expression of SCN5A, only background
vation by 11 mV (468). Current-clamp recordings found levels of SCN10A transcripts were detected in adult human
that A-803467 markedly reduced firing frequency of intra- and mouse hearts (463). However, expression of SCN5A
cardiac neurons (468). The same study showed that may be tuned by multiple cis-regulatory elements located
A-803467 had no effect on the sodium current density and within SCN10A introns (463). A direct correlation of
gating kinetics or action potential waveform in ventricular SCN5A expression was identified with the presence of the
myocytes. In mongrel dogs, direct injection of A-803467 rs6801957 risk-associated SNP in the SCN10A intronic en-
into the anterior right and inferior right cardiac ganglion- hancer in humans, and each additional copy of the minor
ated plexi suppressed the effects of vagus nerve stimulation allele significantly reduced SCN5A expression by 12%
on cardiac conduction (368). Administration of A-803467 (463).
significantly decreased the incidence of AF and shortened
duration and prolonged cycle length of AF (74). A recent The coexpression of WT Nav1.8 with Nav1.5 doubles the
study also showed that selective blockade of Nav1.8 atten- peak sodium current density compared with expression of
uated ischemia-induced ventricular arrhythmia by a mech- Nav1.5 alone; however, replacing WT Nav1.8 with mutant
anism that suppressed the left stellate ganglion (501). Taken channel (R14L and R1268Q) identified in patients with
together, the neuronal hypothesis suggests that Nav1.8 Brugada Syndrome resulted in ~80% reduction of sodium
channels express in intracardiac neurons and exert negative current density (230). The R14L mutation depolarizes
chronotropic and dromotropic effects on sinus node, AV Nav1.8 activation while R1268Q hyperpolarizes Nav1.8
node, and ventricles. steady-state fast inactivation. The association of Nav1.8
and Nav1.5 in cardiac myocytes is supported by coimmu-
3. Cardiomyocyte hypothesis: expression of Nav1.8 noprecipitation studies (230). However, given the prepon-
in cardiac myocytes derance of evidence for a lack of robust expression of
Nav1.8 in cardiac myocytes, the interaction of Nav1.5 and
Nav1.8 transcripts have been reported in mouse and human Nav1.8 in HEK-293 cells reported by Hu et al. (230) may
heart (69, 439, 463, 494). Immunohistochemistry has been not reflect the situation in vivo.

BENNETT ET AL.
F. Targeting Nav1.8 for Pain Treatment 314 to these mutant channels has been attributed to a
relief of steric hindrance or an alteration of microhydro-
Current small-molecule sodium channel blockers in clin- phobicity environment (473). Although QX-314 is not in
ical use share the common feature of lack of isoform clinical use, the differential response of this mutant chan-
selectivity (330). Local anesthetics, such as lidocaine, nel to QX-314 provides proof of principle that a phar-
benzocaine, and tetracaine, have been used as injectable macogenomic approach to treatment of patients carrying
and topical agents for over a century; however, their Nav1.8 mutations might be possible.
analgesic efficacy is accompanied by dose-limiting side-
effects on the CNS, such as seizures, ataxia, confusion,
and sedation (255). V. PAIN AND NaV1.9
A-803467, an inhibitor for Nav1.8 with ⬎100-fold selec- Characterization of Nav1.9 channels has lagged behind
tivity over other Nav isoforms, has provided a proof of other sodium channels because it is difficult to study its
principle that acute and selective pharmacological blockade properties in isolation in native neurons, and until recently
of Nav1.8 can produce significant analgesia in rodent mod- expression of Nav1.9 in heterologous expression systems
els of neuropathic and inflammatory pain such as spinal produced low levels that hampered further studies of this
nerve ligation, sciatic nerve injury, capsaicin-induced sec- channel. Recent genetic and functional findings that link
ondary mechanical allodynia, and thermal hyperalgesia Nav1.9 to human pain disorders (202, 203, 231, 280, 281,
following intraplantar CFA injection (244). Further 343, 508) have reinvigorated research on Nav1.9 as an im-
structural-functional studies revealed that the binding site portant contributor to pain in humans. In this section, we
of A-8034637 partially overlaps with that of tetracaine review studies of Nav1.9, including its pattern of expres-
(58), suggesting that isoform selectivity might be achieved sion, subcellular localization, and modulation, which have
at sites close to that for local anesthetics binding (143). As yielded new insights into the unique gating characteristics
A-803467 has poor solubility and low oral bioavailability of this channel and its role in action potential firing in
in rats, limiting the administration of this agent, modifica- neurons. We also discuss the role of Nav1.9 as an important
tions on the furane nucleus of A-803467 to improve aque- contributor to painful and painless channelopathies in hu-
ous solubility and oral bioavailability also produced anti- mans.
nociception in vivo in rodent models of inflammatory and
neuropathic pain (269, 413). More recently, a novel selec-
tive and orally bioavailable Nav1.8 blocker, PF-01247324, A. Nav1.9 Sequence, Expression Pattern,
was reported to have ⬎50-fold selectivity over other Nav and Subcellular Distribution
isoforms and was shown to reduce excitability in both rat
and human DRG neurons in vitro. This Nav1.8 blocker was Nav1.9 is encoded by the gene SCN11A and was first iden-
reported to alleviate both inflammatory (carrageenan-in- tified in DRG and TRG neurons from rat in which it was
duced thermal hyperalgesia and CFA-induced tactile allo- called NaN or SNS2 (122, 350, 450). SCN11A maps to
dynia) and neuropathic pain (spinal nerve ligation) in ani- human chromosome 3 (3p21–24), a cluster including
mal models in vivo (354). Surprisingly, the structure of SCN5A and SCN10A (the genes encoding Nav1.5 and
PF-01247324 is similar to lamotrigine, a first-generation Nav1.8, respectively), and to the analogous region on
nonselective Nav blocker; thus it is not clear how specificity mouse chromosome 9 (124). The rodent Nav1.9 open-read-
for Nav1.8 is achieved, especially in the absence of a high- ing frame comprises a 1,765-amino acid protein, whereas
resolution atomic three-dimensional structure for the chan- human Nav1.9 is a 1,791-amino acid protein, which retains
nel (255). all of the hallmarks of VGSCs. Human Nav1.9 is only 75%
identical to rodent channels; this is the highest sequence
A recent report showed that QX-314, the membrane- divergence among the rest of the VGSC orthologs (123). For
impermeable charged quaternary amine derivative of li- example, mouse and rat Nav1.6 and Nav1.8 open-reading
docaine, blocked 30% of the peak current (FIGURE 16, frames are 99.5 and 95.3% identical, respectively (365,
A–C) and enhanced use-dependent inhibition of the 440), and mouse and human Nav1.6 amino acid sequences
Nav1.8 gain-of-function mutant channel I1706V from a are 98.6% identical (365). Mouse and rat Nav1.9 channels
patient with painful peripheral neuropathy (FIGURE 16D) are 89% identical at the amino acid level, but only 80%
(234). The I1706 residue is located in a narrow region of identical at the nucleotide level (124), raising the possibility
the pore with its side chain oriented toward the pore, of additional species-specific regulation of these channels at
guarding the extracellular access of local anesthetics (67, the level of RNA processing or codon usage, which might
371). Extracellular application of QX-314 was shown to impact the contribution of the channel to neuronal firing.
block peak currents of Nav1.2 and Nav1.4 channels when
their corresponding isoleucine residue is substituted by The expression of Nav1.9 appears to be tightly regulated, as
smaller amino acid residues, such as alanine, cysteine, or reflected in the narrow expression pattern within the DRG
glutamic acid (371, 444, 473). The accessibility of QX- neurons (FIGURE 17, A–C), which most likely represents

FIGURE 16. Extracellular QX-314 specifically blocks I1706V without affecting wild-type (WT) channel. A:
normalized peak current-membrane voltage (I-V) relationship for activation of WT channels (n ⫽ 6) before
treatment, after 10-min wash-in of vehicle, and after 10-min wash-in of 1 mM QX-314. B: normalized I-V curve
of I1706V channels (n ⫽ 9) before treatment, after 10-min wash-in of vehicle, and after 10-min wash-in of 1
mM QX-314. External QX-314 blocked I1706V currents without significant effect on WT currents. C: nor-
malized peak inward currents of neurons expressing WT (n ⫽ 6) or I1706V (n ⫽ 9) channels in the presence
of vehicle or 1 mM QX-314. Externally applied quaternary blocker QX-314 barely altered the normalized peak
current of WT (P ⫽ 0.871), but it significantly decreased the normalized current of I1706V by ~30% (*P ⬍
0.001). There was no significant difference (P ⫽ 0.991) in the normalized peak current treated by vehicle
between WT and I1706V channels. D: action of externally applied QX-314 on WT (vehicle, n ⫽ 8; QX-314, n
⫽ 6) and I1706V (vehicle, n ⫽ 11; QX-314, n ⫽ 14) channels, respectively, for the time indicated. Currents
were evoked by depolarizing pulses to 0 mV for 50 ms at 10-s intervals for 5 min. The data in each experiment
were normalized with respect to the control currents. [Adapted with permission from Huang et al. (234).]
transcriptional regulation of the channel. Nav1.9 is prefer- to be predominantly present in peptidergic DRG neurons
entially expressed in rodent small-diameter (⬍30 ␮m diam- innervating the colon (219). The expression of Nav1.9 in
eter) DRG and trigeminal neurons (25, 115, 122, 168, 350, sensory and myenteric neurons is supported by molecular,
450), including functionally identified nociceptors (153, immunohistochemical data, as well as the recording of the
154) and intrinsic myenteric neurons (350, 396). Nav1.9 persistent TTX-R current that is produced by this channel.
has also been reported in large-diameter DRG neurons (15,
153, 171). Consistent with expression of Nav1.9 in myelin- The distribution of Nav1.9 in humans is not as well studied
ated fibers, the characteristic Nav1.9 persistent TTX-R cur- as that in rodents because of limited availability of tissues
rent was recorded in a subset of myelinated Ah-type vagal suitable for analyses, but current evidence suggests a similar
afferent neurons (369), and in A␦-type nociceptors (87). pattern of expression in sensory and myenteric neurons.
Nav1.9 has also been found by RNAseq and in situ hybrid- Nav1.9 has been reported in human small-diameter DRG
ization to be enriched in hair cells, which are sensory recep- neurons and their axons (90, 123), and in TRG and axons
tors for auditory and vestibular system, that are marked by innervating dental pulp and teeth (478). Investigation of
the expression of the transcription factor Atoh1 (61). differential expression of Nav1.9 in peptidergic and non-
Within the small-diameter DRG neurons, Nav1.9 is prefer- peptidergic subclasses of human DRG and TRG neurons
entially expressed in somatosensory nonpeptidergic DRG has been hampered because human sensory neurons may
neurons, which can be marked by binding to the isolectin not express the IB4 lectin, which has served as a general
Griffonia simplicifolia IB4 (115), but has also been reported marker for nonpeptidergic neurons (105). The expression

BENNETT ET AL.
of Nav1.9 in the gut tissue in humans has been reported in magnocellular neurosecretory cells in the rodent hypothal-
the myenteric intrinsic primary afferent neurons (340). amus (47). Thus Nav1.9 appears to be primarily expressed
in peripheral sensory and myenteric neurons, but could be
There have been a few reports of the expression of Nav1.9 expressed in focal nuclei in the CNS.
in CNS neurons. Jeong et al. (246) reported widespread
expression in the rat CNS, and O’Brien et al. (339) reported Nav1.9 has been found at all neuronal compartments in
the expression of Nav1.9 in mouse retina, albeit without primary afferents (FIGURE 17, A–C) (118). The channel is
demonstrating the persistent TTX-R current that is attrib- found within DRG neuronal somata, along the nerve fibers
utable to Nav1.9 in these neurons. Another study has re- and free nerve terminals in the epidermis (90, 171, 291,
ported the presence of Nav1.9 in layer V of the medial 358) and central terminals within the outer layers of the
prefrontal cortex in rats using immunohistochemical stain- substantia gelatinosa in the spinal cord (15). It has also been
ing and the prevention of neuronal depolarization in re- found in the majority of unmyelinated fibers innervating the
sponse of cholinergic receptor-mediated stimulation upon cornea, including the bulb-like terminals of the leash fibers
the inclusion of anti-Nav1.9 antibody in the pipette solu- that are present in the most superficial layers of the corneal
tion, albeit without showing the characteristic Nav1.9 per- epithelium (48, 171), and in unmyelinated fibers innervat-
sistent TTX-R current in these neurons (272). Recently, ing the lip skin and dental pulp (350). Although Nav1.9 is
Nav1.9 was reported using immunohistochemical staining predominantly found in unmyelinated C fibers, it has been
and the recording of the characteristic Nav1.9 persistent detected in nociceptive A␦ and A␤ fibers, including at some
TTX-R current in vasopressin- and oxytocin-producing nodes of Ranvier in myelinated fibers (153, 171). Consis-

tent with expression of Nav1.9 in myelinated fibers, the these currents and sensitivity to the composition of the re-
characteristic Nav1.9 persistent TTX-R current was recording buffers; however, protocols have been designed to
corded in a subset of myelinated Ah-type vagal afferent minimize this instability and generate reproducible data
neurons (369). (88, 100, 308, 420). Nav1.9 currents were first recorded in
isolation in DRG neurons from mice in which Nav1.8, the
other major TTX-R channel, was knocked out (100); the
B. Biophysical Properties of Nav1.9
TTX-S currents that might have been present in these neu-
Channels
rons were blocked with 500 nM TTX in the bath solution.
These recordings showed that Nav1.9 channels characteris-
Nav1.9 channels produce a TTX-R sodium current with
tically activate at hyperpolarized voltages, compared with
gating properties that distinguish them from those pro-
other neuronal sodium channels, and inactivate with un-
duced by other members of this family (FIGURE 17, D AND
E), such that Nav1.9 currents could be identified in neurons
usually “ultra-slow” kinetics, which results in the persis-
that produce multiple sodium channels (118). tence of the sodium current after activation (100) (FIGURE
17, D AND E). More extensive glycosylation of Nav1.9 is
Recordings of sodium currents in DRG neurons from observed in neonatal compared with adult DRG neurons
Nav1.9 knockout mice show that the persistent TTX-R cur- (FIGURE 17F), which is paralleled by a hyperpolarized volt-
rent is absent (15, 349, 367), consistent with the attribution age dependence of inactivation, but not activation (FIGURE
of this current to Nav1.9. More direct evidence that Nav1.9 17G) (460). Pretreatment of neonatal DRG neurons with
channels produce the persistent TTX-R current came from neuraminidase results in Nav1.9 currents that are indistin-
studies showing the restoration of this current by the ex- guishable from those recorded in untreated adult DRG neu-
pression of recombinant Nav1.9 channels in DRG neurons rons. It should be noted, however, that the hyperpolarized
from the Nav1.9 knockout mice (231, 349). Recombinant activation of Nav1.9 is more prominent in pipette solutions
Nav1.9 currents have now been convincingly expressed and that contain fluoride compared with chloride-based inter-
studied in isolation in cell types that do not express endog- nal solution (88, 420). The fluoride-induced hyperpolariz-
enous TTX-R sodium currents, including rat SCG neurons ing shift in Nav1.9 activation permits the isolation of this
(202, 203), the DRG-derived cell line ND7/23 (280, 281, current by digital subtraction of TTX-R current recorded
464), and HEK-293 cells (287). using a holding potential of ⫺60 mV, where the current is
primarily produced by Nav1.8 channels, from the total
Studying biophysical properties of Nav1.9 currents has TTX-R current recorded with a holding potential of ⫺120
been challenging because of widely reported instability of mV (100). The large overlap between activation and inac-
FIGURE 17. Distribution of Nav1.9 in primary afferents, glycosylation, and biophysical properties. A: voltage-gated sodium channel 1.9
(Nav1.9; red) is present in most protein gene product 9.5 (PGP9.5)-positive (green) intraepidermal nerve fibers, which have endings in the
epidermis that extend from the subepidermal nerve bundle. Colocalization of Nav1.9 and PGP9.5 is yellow. The dotted line indicates demarcation
of stratum corneum (above the line) and stratum lucidum (below the line) and of the epidermis. The different layers of the epidermis are indicated
by brackets (Sb, stratum basale; Ss, stratum spinosum; Sg, stratum granulosum; Sl, stratum lucidum; Sc, stratum corneum). B: small-diameter
neurons in the dorsal root ganglia (DRG) exhibit robust Nav1.9 immunolabeling (red). DRG neurons display substantial colocalization of Nav1.9
with the Griffonia simplicifolia isolectin IB4 (green), but not with calcitonin gene-related peptide (CGRP; blue). C: presynaptic nerve terminals of
DRG neurons in the superficial layers of the dorsal horn of spinal cord display Nav1.9 immunostaining (red). The dorsal horn is outlined by dotted
line. D: the persistent TTX-R (resistant) sodium currents mediated by Nav1.9 channels and, for comparison, fast-inactivating TTX-S (sensitive)
currents mediated by Nav1.6 and/or Nav1.7 channels recorded from a small-diameter Nav1.8-null mouse DRG neuron that lacks the slow TTX-R
current mediated by Nav1.8 channels. Current traces that result from 100-ms depolarizing pulses from a holding potential of ⫺120 mV (to
ensure that all Nav channels are available to open) to the membrane voltages indicated on the traces show that Nav1.9 channels activate at more
hyperpolarized potentials than do the fast TTX-S channels. Depolarization to ⫺40 mV activates only Nav1.9 channels (blue trace), whereas a
depolarizing stimulus at ⫺30 mV shows a compound trace consistent with activation of both a fast-inactivating TTX-S component (magenta
trace) and the Nav1.9 persistent component (blue trace). Note the rapid activation and inactivation of the TTX-S current, compared with the slow
activation and very slow inactivation of Nav1.9. E: fitting the activation data of Nav1.9 currents (solid maroon curve) and TTX-S currents (solid
blue curve) to Boltzmann’s function shows that Nav1.9 channels activate at more hyperpolarized potentials (that is, they open more easily at
hyperpolarized potentials) than do the fast-activating and -inactivating TTX-S sodium channels. The steady-state inactivation curves for TTX-R,
Nav1.9 persistent channels (dashed maroon line), and fast-inactivating TTX-S sodium channels (dashed blue line symbols) show that Nav1.9
channels remain available at more depolarized potentials than do the TTX-S channels. The voltage domain delineated by the overlap between
activation and inactivation curves correspond to the “window current” that is produced by the different channels and is substantially larger for
Nav1.9 (maroon) than for the TTX-S channels (blue). Window currents of Nav1.9-expressing DRG neurons are predicted to induce depolarization
of resting membrane potential and thus contribute to boosting weak stimuli. F: DRG membrane fractions from day of birth (P0) and postnatal
day 7 (P7) rats contain two immunoreactive proteins. Trace amount of the slower migrating band is present in embryonic day 17 (E17) sample,
while postnatal day 21 (P21) DRG sample contains only the faster migrating species. The slow-migrating protein is a glycosylated form of Nav1.9.
G: steady-state voltage dependence of inactivation of neonatal, adult, neonatal desialidated, and adult desialidated Nav1.9 channels. Treatment
of DRG neurons with neuraminidase causes a depolarizing shift of steady-state inactivation of Nav1.9 currents in neonatal neurons, but not adult
neurons. [A–E are used with permission from Dib-Hajj et al. (118); G and H are adapted with permission from Tyrrell et al. (460).]

BENNETT ET AL.
tivation of Nav1.9 results in a big window current within neuronal cell types reported to date (202, 203, 280, 281,
the physiological voltage domain close to RMP of DRG 464), however, show that activation of recombinant human
neurons (⫺70 mV to ⫺40 mV, FIGURE 17E). Nav1.9 is comparable to that of native currents in rodent
DRG neurons. Activation of recombinant rodent Nav1.9
Another level of regulation of Nav1.9 reflects the formation channels is hyperpolarized by 12–14 mV, compared with
of functional channel complexes and delivery to the plasma recombinant human Nav1.9, when the channels are ex-
membrane. Thus the initial failure to express recombinant pressed in HEK-293 cells (287). The discrepancy between
Nav1.9 channels in mammalian cell lines such as HEK-293 these data could be explained by differences in the back-
cells (115, 349) may reflect the lack of factor(s) that are ground of the cells used in the different assays, the small
essential for forming functional channels. Using the yeast number of human native neurons that were studied in the
two-hybrid genetic approach followed by biochemical val- Dib-Hajj et al. study (123), or the possibility that human
idation, a member of the fibroblast growth factor family DRG neurons include multiple species of Nav1.9 channels
(FGF12, also known as FHF1) was found to interact with with different primary sequence, or modifications of the
the COOH-terminus of Nav1.9 (290). Using an affinity pu- Nav1.9 channels that are different in rodent and human
rification approach followed by immunohistochemical and DRG neurons. More data, especially from human native
biochemical validation, the cell adhesion molecule contac- DRG neurons, are needed to distinguish between these dif-
tin was also found to interact with the COOH-terminus of ferent hypotheses.
Nav1.9 and colocalize with the channel in rat small DRG
neurons, extending to nerve endings in the skin, and in- Differentiated sensory neurons derived from iPSCs could
creases the channel density at the cell surface of Chinese become another source of neurons to study properties of
hamster ovary cells (291). The current density of the Nav1.9 Nav1.9 in human cells. Recent studies have reported a
persistent TTX-R current was reduced in DRG neurons mixed picture regarding the expression of Nav1.9 in iPSC-
from a contactin-null mouse (398), in agreement with bio- derived sensory neurons. Although Chambers et al. (70)
chemical finding in the heterologous expression system. reported the presence of Nav1.8 TTX-R currents in their
However, the coexpression of FHF1 or contactin with differentiated sensory neurons, they did not report the ex-
pression of Nav1.9. Two studies that used differentiation
Nav1.9 did not boost the current levels in mammalian cell
protocols that were modified from the one employed by
lines above background levels, as did the coexpression of
Chambers et al. also did not detect Nav1.9 currents in these
sodium channel ␤-subunits, suggesting that additional fac-
neurons, despite the detection of transcripts of this channel
tors are necessary to form a functional Nav1.9 channel com-
(134, 500). By contrast, two studies that used a different
plex (S. D. Dib-Hajj, B. Tanaka, D. Sizova, T. R. Cummins,
differentiation paradigm (52, 471) recorded TTX-R cur-
and S. G. Waxman, unpublished observations). A recent
rents from sensory neurons transdifferentiated from fibro-
study, however, has shown that Nav1.9 chimera in which
blasts; however, only one of them showed traces that were
the COOH-terminus of the channel was replaced with that
consistent with a Nav1.9 current, but were of a small mag-
of the Nav1.4 channels produced currents in HEK-293 cells
nitude (471). Thus, while there is the promise of using iPSC-
with activation properties that are similar to the Nav1.9
derived sensory neurons as a source material to study prop-
parent channel in neuronal cells, but with accelerated inac- erties of human Nav1.9 channels, it appears that protocols
tivation properties (191), adding to the evidence that the that produce more robust currents are needed for more
COOH-terminus of the channel is necessary either to bind extensive studies of this channel.
to an auxiliary channel subunit that is essential for forming
a functional channel complex at the cell membrane, or for 1. Nav1.9 as a threshold channel
the trafficking of the channel from cytoplasmic pools to the
cell membrane. The more recent study by Lin et al. (287) The role of Nav1.9 as a threshold channel for action poten-
reporting the expression of full-length WT human, rat, and tial electrogenesis is supported by anatomical and func-
mouse Nav1.9 channels in HEK-293 cells would indicate tional data, as well as computer simulations. As shown in
that the missing factor(s) in these earlier studies can be FIGURE 17, Nav1.9 is located along the entire trajectory of
expressed in some isolates of HEK-293 cells. primary afferents from the peripheral receptor fields to the
presynaptic terminals within the dorsal horn. The ultra-
Initial recordings from human native DRG neurons re- slow kinetics suggest that Nav1.9 channels do not contrib-
ported that Nav1.9 channels activate at approximately ⫺80 ute much to the amplitude of action potentials, but the large
mV, 10 –20 mV more negative than native Nav1.9 currents window current bracketing the voltage domain around the
in rodent DRG neurons, and this difference was interpreted physiological resting potential of neurons, and hyperpolar-
to be due to species-specific differences in primary protein ized activation relative to the other sodium channels, sug-
sequence (123). The hyperpolarized activation voltage in- gest that Nav1.9 has a robust depolarizing effect on resting
dicates that human Nav1.9 can open in response to a potential and can act as a threshold channel (100). Com-
weaker stimulus compared with rodent Nav1.9. Activation puter simulations show that, even at 50% of its normal
and inactivation of human Nav1.9 channels in the different density in DRG neuron cell bodies, Nav1.9 contributes

⬎75% of its depolarizing effect (215). By analogy to mu- D. Nav1.9 in Rodent Models of Pain
tant Nav1.7 channels from patients with IEM, which evoke
DRG neuron hyperexcitability, both by depolarizing rest- Initial studies in Nav1.9-null mice reported normal acute
ing potential and via an increase in sodium conductance mechanical and thermal pain thresholds (15, 283, 367).
(209, 466), the reduction in action potential threshold pro- These findings were surprising, since the experimental evi-
duced by Nav1.9 is likely caused both by depolarization of dence supporting a role for Nav1.9 as a threshold channel is
the resting potential and by the increase in sodium conduc- strong. Subsequent studies have since shown that Nav1.9
tance that it imposes on the cell membrane. Empirical evi- knockout mice can manifest a deficit in mechanical pain
dence supporting a role for Nav1.9 as a threshold channel thresholds when the stimulus is presented at a different
was provided by experiments in which it was shown that a location, tail compared with hindpaw (315), or when the
GTP-mediated upregulation of the Nav1.9 current lowers thermal and mechanical stimuli were presented as slow
the current threshold for action potential generation in ramps rather than the conventional steplike application,
DRG neurons, increases repetitive firing, and can drive and this has been interpreted to reflect the slow kinetics of
spontaneous activity (20, 349). Similarly, Nav1.9 channels the Nav1.9 channel (227). The molecular and cellular bases
were shown to regulate threshold for action potential firing for these findings are unclear at present.
in myenteric neurons (86, 347).
Animal studies, however, support a role for Nav1.9 in sev-
eral induced pain models. Nav1.9 has been shown to con-
C. Regulation of Nav1.9 by Trophic Factors, tribute to inflammatory pain (15, 298, 367) following the
Cytokines, and Inflammatory Mediators injection of melittin (502). Nav1.9 has been shown to be
upregulated in large-diameter DRG neurons in the STZ
model of diabetes in rats (93) and has been implicated in
Nav1.9 expression is regulated by the trophic factor GDNF,
bone cancer pain (370) and cold pain (299). Nav1.9 has also
but not NGF (168, 279), although the signal cascade down-
been shown to play a role in visceral pain (307, 385).
stream of the receptors for this factor that targets the chan-
nel is not well understood. The inclusion of GTP in the
Studies using nerve transection in rats to assess effects of
recording pipette causes a significant upregulation of the
Nav1.9 on pain did not yield conclusive evidence support-
current density of Nav1.9 (19, 20, 349), indicative of mod- ing a role for this channel in neuropathic pain. Transection
ulation of the channel activity or density of channels at the of the sciatic nerve (axotomy at midthigh level), or in one of
cell surface by GTP-binding proteins. Nav1.9 current den- its branches (spared injury nerve model) leads to marked
sity is increased by treatment with inflammatory mediators reduction of Nav1.9 mRNA and protein levels and the
and their downstream signaling cascades (19, 20, 35, 219, TTX-R persistent current density (99, 104, 107, 122, 434),
303, 349, 401). PGE2, an inflammatory pain mediator suggesting that this channel may not play an important role
(361), increases the Nav1.9 current in DRG neurons via a in neuropathic pain. It should be noted that these traumatic
pathway that involves G proteins (401), but may require nerve injury animal experiments are typically done by ligat-
chronic exposure to manifest its effects, since acute appli- ing the transected nerves, thus preventing trophic support
cation does not lead to alteration in the Nav1.9 current from the previously innervated tissues from reaching the
(303, 513). Treatment with a well-defined mixture of in- transected fibers. Indeed, intrathecal delivery of the GDNF
flammatory mediators, bradykinin, ATP, histamine, PGE2, rescues the expression of Nav1.9 channels in DRG neurons
and norepinephrine, but not when its components are ap- (99) and partially restores the conduction velocity of C
plied individually, causes a marked increase in the current fibers in axotomized animals (26). Another axotomy model
density of Nav1.9 and enhanced firing of DRG neurons that involved simple transection of the nerve, which may
from WT but not Nav1.9-null mice (303). Application of a have been associated with more inflammation than the in-
similar mixture of inflammatory mediators, bradykinin, jury models described above, and which would certainly
ATP, histamine, PGE2, and 5-hydroxytryptamine, causes allow access of cellular factors from the surrounding tissues
enhanced firing of afferents that innervate the colons of WT to the transected fibers, showed an increase in TTX-R cur-
mice, but not Nav1.9-null mice (219). Interestingly, activa- rents, including those attributable to Nav1.9 (1). Thus sur-
tion of the neurokinin-3 (NK3) receptor in myenteric neu- gically transected nerves in humans, which are typically
rons increase Nav1.9 current density due to a rapid, tran- embedded in muscle or other tissues where the cell bodies
sient hyperpolarizing shift of activation and inactivation, an may receive trophic factors, could lead to the upregulation
effect that is mimicked by activation of PKC (86). NK3 has of Nav1.9 channels and contribute to neuropathic pain.
been implicated in visceral pain following colorectal disten-
tion and visceral hypersensitivity induced by stress and has The studies in the rat are paralleled by the absence of pain
been shown to increase excitability of C fibers (408). These deficits following sciatic nerve injury in Nav1.9 knockout
findings support a role for this channel in rendering DRG mice (15, 367). Another independent Nav1.9 knockout
neurons hyperexcitable in inflammatory disorders or pa- strain (349) did not impair mechanical or thermal pain
thologies, where proinflammatory cytokines are produced. following a second-degree burn injury (431). The knock-

BENNETT ET AL.
down of Nav1.9 using antisense ODN treatment did not genetic differences that may have introduced unintended
ameliorate pain in rats (366), and thus it remains to be seen variables into the testing of these mice.
whether Nav1.9 could contribute to neuropathic pain.
However, these results may have been confounded by com-
pensatory change in knockout mice that masks the effect of E. Nav1.9 Variants in Human Pain Disorders
this channel on neuropathic pain, and the knockdown ex-
periments have not been independently verified. It has been The discovery and characterization of monogenic links of
reported, on the other hand, that cold pain is impaired in mutations in Nav1.9 and human pain disorders (TABLE 6)
Nav1.9-null mice following CCI to the sciatic nerve (283). has validated the role for this channel in human pain, de-
By analogy to the data on acute pain thresholds, however, it spite the reported discrepancies in the rodent models. Mu-
tations in Nav1.9 (FIGURE 18 AND TABLE 6) have been iden-
is not known whether testing pain behavior at locations
tified in individuals with rare pain diseases and in individ-
other than the hindpaw or by using ramplike stimuli might
uals with the more common painful peripheral neuropathy.
unmask deficits in neuropathic pain in Nav1.9 knockout
Dominant gain-of-function mutations in Nav1.9 have been
mice.
found in individuals with inherited pain disorder with onset
in early childhood (203, 280, 282, 343, 508) or adult-onset
The preponderance of evidence supports a role for Nav1.9
painful peripheral neuropathy (202, 231), and in individu-
in inflammatory pain in animal models, consistent with the
als with the inability to experience somatic pain associated
effect of inflammatory mediators on Nav1.9 current density
with abdominal pain and gastrointestinal dysfunction (281,
and on Nav1.9-mediated increase in DRG neuronal excit-
483). These studies have validated the Nav1.9 channel as a
ability (see section above). Nav1.9 knockout mice show
contributor to human pain, albeit with a complicated mech-
impaired mechanical allodynia and thermal hyperalgesia anism for causing both hyperexcitability and hypoexcitabil-
following somatic inflammation (15, 367). Priest et al. ity of DRG neurons, as will be discussed below.
(367) showed that mice carrying one null-allele manifest
impaired response, consistent with haploinsufficiency. Al-
1. Familial episodic pain
though mice that are heterozygous for the null-allele
showed an impaired response in some of the tests performed
Familial episodic pain linked to mutations in Nav1.9 have
on a different Nav1.9 knockout mouse, the data did not
been reported in three Chinese families (282, 508), one
always reach statistical significance (15). Thus it is not clear
family of mixed European ancestry (280), one family from
whether Nav1.9 haploinsufficiency is a common feature in
South America (203), and six families from Japan (343).
these transgenic mice. Another independently produced
Common features of these cases are as follows: early onset
Nav1.9 knockout mouse, however, only partially repro- in childhood and symptoms mainly in lower extremities and
duced the inflammatory pain deficits reported in these ear- joints. Fatigue and cold were triggers for several of these
lier studies (283). It is not entirely clear what causes the patients, and pain was alleviated by warming extremities
discrepancies in the behavior of these Nav1.9 knockout and applying compressors (280, 343, 508). Leng et al. (282)
mouse strains. reported adult-onset essential tremor in affected individu-
als, after the cessation of pain episodes, with no other sen-
The enhanced firing of afferents that innervate the colon of sory deficits suggestive of peripheral neuropathy, whereas
WT mice, but not Nav1.9-null mice, when treated with an Zhang et al. (508) did not report this symptom in one of the
inflammatory soup (219), and the effect of NK3 on increas- families that carry the same mutation (Arg225Cys). By con-
ing Nav1.9 current density and firing of DRG neurons trast, pain attacks were mostly not associated with identi-
(303), suggest a role for this channel in visceral pain. Earlier fiable triggers, although occasionally they were triggered by
studies using Nav1.9 knockout mice, however, did not man- high ambient temperatures in the family from South Amer-
ifest gastrointestinal or apparent nutritional deficits (15, ica, and pain relief could be achieved by cooling affected
367). Using the same strain of mice that was reported by limbs or applying pressure (203). Pain relief was also
Priest et al. (367), it has been shown that Nav1.9 channels achieved by treatment with nonsteroidal anti-inflammatory
contribute to symptoms of urinary bladder inflammation drugs (NSAIDs) in these patients except for those from the
(385). Leo et al. (283) reported enhanced writhing behavior South America family.
in response to acetic acid challenge in these knockout mice,
suggesting a surprising antinociceptive role for the Nav1.9 Genetic linkage and sequence analyses identified five muta-
channels in visceral neurons. Electrophysiological record- tions in Nav1.9 (FIGURE 18 AND TABLE 6), all affecting
ings from DRG neurons innervating the peritoneum follow- residues that are conserved in all human sodium channel
ing visceral inflammatory in these mice, however, did not isoforms. Three mutations altered a residue within the volt-
manifest impaired excitability (216). These discrepancies in age-sensor segment 4 of domain I (DI–S4; R222H, R222S,
the outcomes of animal studies using the different strains of and R225C), one mutation substituted a residue within the
Nav1.9 knockout mice may reflect interlaboratory differ- pore-lining segment 6 of domain II (DII–S6; A808G), and
ences in methodologies and, importantly, genomic and epi- one mutation substituted a residue in the pore-lining seg-

TABLE 6. Summary of published mutations linked to painful and painless Nav1.9 channelopathy showing the electrophysiological characteristics
Suprathreshold
Response
to
V1/2 Slow Persistent Spontaneous Current Current
Amino Acid Pubmed ID Nucleotide V1/2 act inact Repriming Inactivation Deactivation Resurgent Ramp Current Activity RMP Threshold Injection
R222H 27503742 665G⬎A Hyperpol UC NT UC UC NT UC UC Increased Depol Reduced Increased

R222H 27224030 665G⬎A UC NT NT NT NT NT NT NT NT NT NT NT
R222S* 27224030 664C⬎A UC NT NT NT NT NT NT Reduced Increased UC Reduced Increased
R225C 24207120 673C⬎T UC UC NT NT NT NT UC UC Increased UC NT Increased
I381T 24776970 1142T⬎C Hyperpol Depol NT UC Decrease NT NT UC Increased Depol Reduced Increased
G699R 25791876 2095G⬎A Hyperpol Depol NT UC Decrease NT Reduced UC UC Depol Reduced Increased
L799P† 24036948 Hyperpol Hyperpol NT NT Decrease NT NT Increased NT Depol NT NT
A808G 24207120 2423C⬎G UC UC NT NT NT NT UC UC Increased UC NT Increased
L811P 24036948 2432T⬎C Hyperpol UC NT NT Decrease NT NT Increased NT Depol NT Increased
L811P 26645915 2432T⬎C NT NT NT NT NT NT NT NT NT Depol Increased
L811P§ In press 2432T⬎C NT NT NT NT NT NT NT NT UC‡ Depol UC‡ NT
L1158P 24776970 3473T⬎C Hyperpol UC NT UC Decrease NT NT Increased UC Depol Reduced Increased
N1169S 27781142 3506A⬎G NT NT NT NT NT NT NT NT NT NT NT NT
V1184A 26645915 3551T⬎C Hyperpol UC NT NT Decrease NT NT UC NT Depol NT Increased

I1293V 27781142 3877A⬎G NT NT NT NT NT NT NT NT NT NT NT NT
L1302F 26746779 3904C⬎T NT NT NT NT NT NT NT NT NT NT NT NT
L1302F In press 3904C⬎T Hyperpol UC NT NT NT NT NT UC UC‡ Depol UC‡ NT

inactivation, respectively. *R222S: Current-clamp analysis was done in dorsal root ganglia neurons from a knock-in mouse. †L799P: Substitution in native mouse Nav1.7, which
corresponds to L811P in the human channel. ‡UC: Several neurons that were silent at their native very depolarized RMP either fired spontaneously or had very low threshold (⬍110

pA) when cells were held at ⫺60 mV. §L811P: Current-clamp recordings were done in adult rat dorsal root ganglia neurons, as described in Huang et al. (232).
1127
BENNETT ET AL.
FIGURE 18. Location of pain-associated SCN11A mutations. Nav1.9 ␣-subunit structure shows the location
of amino acids associated with human pain disorders.
ment 5 of domain III (DIII-S5; V1184A). These mutations sity or gating properties of the mutant channels R222H/S,
cosegregated with pain in affected individuals, were absent R225C, or A808G. The absence of gain-of-function attributes
from unaffected individuals, and were not present in con- in the voltage-clamp recordings in these studies (343, 508)
trol data sets. may have been a result of methodological issues that masked
the true effect of these mutations on the Nav1.9 channel. By
Okuda et al. (343) investigated the mechanistic basis for contrast, expression of the Arg222His in SCG neurons (203)
Nav1.9-related pain using a transgenic mouse in which they and V1184A in ND7/23 cells (280) revealed that these muta-
knocked in the R222S mutation in the gene encoding mouse tions alter current density and gating properties of the Nav1.9
Nav1.9. Behavioral testing was done at two ages (8 –9 wk channel. Han et al. (203) did not find a significant difference in
and 36 –38 wk), and compared with WT littermates. The the current density (FIGURE 19, A–C), but reported a significant
authors reported no significant difference in mechanical shift of ⫺6.4 mV in the half-maximal voltage of activation and
thresholds, but reduced thresholds for thermal and cold
a larger slope factor for the R222H mutant channel (FIGURE
pain. Although the authors reported different thresholds
19D), consistent with more channels opening at hyperpolar-
of the three sensory modalities at the younger (8 –9 wk)
ized voltages compared with WT channels, and an increase in
and older (36 –38 wk) ages in the transgenic mice, they
did not report whether these pain thresholds differed the window current. Mutant channels also activated faster
significantly with age. Cold pain hyperalgesia of the than WT channels. Leipold et al. (280) recorded Nav1.9 cur-
transgenic mouse parallels the cold-induced pain in pa- rents at 30°C and 20°C to probe the behavior of the channel at
tients carrying this mutation and is consistent with the warm and cooler temperatures. They reported that tempera-
role of Nav1.9 in cold pain (299). It is noteworthy, how- ture did not change the current density of either the WT or the
ever, that the reduction in thermal pain thresholds of this V1184A mutant channel, but reported a twofold increase in
transgenic mouse is incongruent with the use of warmth the current density for the V1184A mutant channel compared
to alleviate pain in patients carrying four of the muta- with WT channels at voltages more negative than ⫺40 mV at
tions in Nav1.9 (280, 343, 508). The authors did not
both recording temperatures. They also reported a large shift
report gastrointestinal deficits in the knock-in mouse, but
of ⫺17.5 mV in the half-maximal voltage of activation at
it is not clear whether they investigated these features in
their published study. 30°C and ⫺19.5 mV at 20°C. However, there were no marked
differences between WT and mutant channels kinetics of
It has been reported that functional testing in either mouse opening and closing at either temperature. The latter two stud-
DRG neurons or ND7/23 heterologous expression system ies clearly demonstrate gain-of-function attributes of the mu-
(343, 508) did not reveal significant differences in current den- tant Nav1.9 channels.

FIGURE 19. Effects of R222H on properties of Nav1.9 channels. Average whole cell sodium currents
recorded from superior cervical ganglion neurons expressing wild-type (WT; A) or R222H (B) Nav1.9 channels
are shown. Holding potential was ⫺120 mV. The displayed current traces have been normalized to the
maximum current amplitude. Blue trace in A and gold trace in B indicate whole cell currents recorded during
test depolarizations to ⫺40 mV. C: average current-voltage relationships determined from cells expressing WT
(open circles) or R222H (orange circles) channels. Current values were normalized to cell capacitance and
show reduced current density of mutant channel, albeit not reaching statistical significance. Data points in C
represent means ⫾ SE. Effects of R222H on voltage dependences of activation and inactivation are shown. D:
superimposed steady-state inactivation and activation curves calculated for WT (black) and R222H (orange)
sodium currents. Steady-state inactivation and activation curves for WT intersect near ⫺46 mV, whereas
those for R222H intersect at approximately ⫺50 mV. Shading indicates window current between ⫺100 and
⫺10 mV. The yellow shaded area represents the window current for R222H channels, and the gray area is the
window current for WT channels. [Adapted with permission from Han et al. (203).]
The expression of the five mutant Nav1.9 channels in tant channels (343, 508). Leipold et al. (280) reported a
DRG neurons resulted in enhanced evoked excitability significant depolarization of the RMP of neurons ex-
with reduced current threshold for action potential and pressing the V1184A mutant channel by ⫹5.8 mV at
higher firing frequency (203, 280, 343, 508). Han et al. 30°C and ⫹5.2 mV at 20°C, and reported that expression
(203) reported significant depolarization of ⫹4 mV of the of the V1148A mutant channel in DRG neurons, com-
RMP of DRG neurons expressing the R222H mutant pared with the expression of WT channels, led to an
channels at 23°C. Depolarization of RMP of DRG neu- enhanced evoked firing during the application of short
rons by a similar magnitude has been previously shown duration stimuli (2 s) and relatively longer stimuli (30 s).
to increase excitability of neurons that express mutant They also reported that cells expressing the mutant chan-
sodium channels (150, 151, 199, 202, 203, 231, 400). In nel retained a higher firing frequency when the cells were
agreement with these data, Han et al. (203) showed that recorded at 20°C, compared with those expressing WT
the expression of the R222H mutant channel reduced channels. This latter result is consistent with the cold-
threshold for the first action potential by one-half, in-
aggravated pain symptoms in the patients carrying this
creased evoked firing in response to a wide range of stim-
uli (FIGURE 20, A AND B), and increased the fraction of mutation.
neurons that fired spontaneously (FIGURE 20C). These
data are consistent with the pain phenotype in this fam- The segregation of symptoms with the presence of the mu-
ily. It is likely that future studies of the R222S, R225C, tation in these families, together with the reported gain-of-
and A808G mutations will show that they confer gain- function attributes at the channel level and the mutant-
of-function attributes on the channel, in line with the channel-induced neuronal hyperexcitability, support a
hyperexcitability of DRG neurons that express these mu- causal link between these Nav1.9 mutations and the com-

BENNETT ET AL.
FIGURE 20. Effects of R222H on resting membrane potential and firing properties of dorsal root ganglia
(DRG) neurons. A: responses of representative DRG neurons expressing wild-type (WT) and R222H channels,
respectively, to 500-ms depolarization current steps, which are 1⫻ and 2⫻ the current threshold for action
potential generation for these two neurons. B: comparison of repetitive action potential firing spike no. between
DRG neurons expressing WT and R222H across a range of 500-ms step current injections from 25 to 500
pA. *P ⬍ 0.05. C: representative recording of spontaneous firing in a DRG neuron expressing R222H mutant
channels. Trace was recorded for 30 s without current injection. Inset: bar graph showing the proportion of
spontaneous firing cells for DRG neurons expressing R222H (red) is 2.8-fold compared with that of WT (black);
numbers to the right of the bar graph show percentage of neurons firing spontaneously for WT (18%) and
R222H (51%). [From Han et al. (203) with permission.]
plex pain phenotype in these patients. By contrast, the started in adulthood. Genetic studies of this cohort identi-
mechanistic basis for the gastrointestinal symptoms in these fied 12 individuals carrying eight mutations in Nav1.9 and
patients and the divergence in the triggers for pain attacks no mutations in Nav1.7 or Nav1.8. Four of these mutations
are not known. in Nav1.9 substitute highly conserved amino acids in trans-
membrane segments of the channel; three substituted resi-
2. Nav1.9 and peripheral neuropathy dues in the cytoplasmic loops or of the carboxyl-terminus of
the channel; and one affected the 3= splice acceptor site of
The contribution of Nav1.9 to more common human pain intron 24. Since these were singleton cases, establishing
disorders is supported by studies that identified and charac- pathogenic potential of these mutations required functional
terized mutations in this channel in a cohort of 345 individ- testing to demonstrate an effect on neurons that could pro-
uals with painful peripheral neuropathy (202, 231). These vide a mechanistic explanation for the clinical symptoms
patients experienced numbness, tingling, and pain in the (476).
distal extremities and diarrhea and complained of dry eyes.
Unlike Nav1.9-related familial episodic pain with onset in Functional assessment of three of these mutations (I381T
infancy as described above, symptoms in these patients in DI–S6, G699R in DII/S4 –5, and L1158P in DIII–S4)

using whole cell voltage-clamp recordings in mouse ulcers at posterior cervical location, slow wound healing,
Nav1.9 knockout DRG neurons (231) or in SCG neurons pruritus, Charcot arthropathy, and diarrhea; these pa-
(202) demonstrated gain-of-function changes in mutant tients also reported intolerance to moderate hot/cold
channels (202, 231). The three mutations caused a sig- weather (232, 281, 363, 483). Other symptoms were
nificant hyperpolarizing shift in the half-maximal voltage private to some affected individuals, including failure to
of activation, which ranged from ⫺6.7 mV to ⫺10.1 mV, thrive secondary to intestinal dysmotility, constipation,
and slowed deactivation of these mutant channels. The episodic abdominal pain, rectal pain upon urination and
I381T and G699R mutations also depolarized the half- defecation, excessive sweating, muscle weakness, and
maximal voltage of inactivation by 13.3 mV and 6.3 mV, joint hypermobility. These patients manifested normal
respectively. The hyperpolarized shift of activation and intellectual ability and normal conduction velocity of pe-
depolarized shift of inactivation markedly increase win- ripheral nerves, functional magnetic resonance brain im-
dow currents produced by these channels, and slower aging, and structural features on sural nerve biopsy in the
deactivation increases the open time of the mutant chan- cases where these tests were conducted. These findings
nels. In addition to the hyperpolarized shift in activation, indicate that the pain insensitivity is not related to faulty
L1158P channels activated with faster kinetics compared sensory neuron wiring, gross abnormality in brain struc-
with WT channels. ture, or intellectual disability. Whole exome sequencing
on affected and unaffected blood relatives in one study
The gain-of-function attributes that these three mutations (281) or targeted sequencing of the SCN11A in the other
conferred on the Nav1.9 channel predicted enhanced excit- studies (232, 363, 483) identified two novel mutations,
ability of DRG neurons that express these channels. Con- L811P in DII–S6 (281, 483) and L1302F in DIII-S6 (232,
sistent with this prediction, current-clamp recordings of rat 363). The L811P mutation appeared de novo in the re-
DRG neurons demonstrated that the mutant channels con- ported carriers, while the L1302F mutation was trans-
fer hyperexcitability, compared with WT Nav1.9 channels. mitted across generations. In contrast to the CIP pheno-
DRG neurons that expressed each of the mutant channels type caused by mutations in Nav1.7, which required bi-
possessed a RMP that was depolarized by 3.5– 4.5 mV,
allelic mutations in SCN9A (see sect. IIIB1), the painless
reduced threshold for action potential firing, increased
phenotype in these individuals is dominant, requiring
number of neurons firing spontaneously, and enhanced fre-
only one allele of SCN11A to carry the mutation.
quency of evoked firing. These current-clamp studies, how-
ever, produce supraphysiological levels of the channels un-
Leipold et al. (281) investigated the mechanistic basis for
der investigation and do not control the level of recombi-
Nav1.9-related insensitivity to pain using a transgenic
nant-channel expression in individual neurons, thus
mouse in which they knocked in the corresponding muta-
requiring relatively large number of neurons to draw mean-
tion in the gene encoding mouse Nav1.9 (L799P). The trans-
ingful conclusions. The development of a dynamic clamp
genic mouse did not manifest morphological changes in
approach permitted assessment of the effect of precisely
cutaneous sensory fibers. Functional testing in a slice prep-
controlled levels of specific sodium currents, modeled after
aration from this transgenic mouse by recording of minia-
WT and mutant sodium channels, on excitability studies in
native DRG neurons (466). Dynamic clamp experiments ture excitatory postsynaptic currents (mEPSCs) showed im-
using a mixed sodium current composed of equal fractions paired transmission into the dorsal horn, suggesting a pos-
of WT and I381T Nav1.9 currents to mimic the heterozy- sible mechanistic explanation for the insensitivity to pain.
gous state in the patients revealed a substantial depolariza- Behavioral assessment of the knock-in mutant mouse, how-
tion of RMP and induced hyperexcitability of DRG neu- ever, showed higher thresholds for noxious heat, and nor-
rons, consistent with the findings from the studies using mal thresholds for plantar acute mechanical and thermal
transfected neurons (231). On the bases of these functional stimulation. These results do not recapitulate the intoler-
observations, these mutations meet the criteria for “patho- ance of patients carrying the L811P mutation to moderate
genic” or “probably pathogenic” substitutions (476). The hot or cold weather. The transgenic mice did show small
rest of the mutations that were reported by Huang et al. but statistically significant higher thresholds for plantar me-
(231) remain of unknown clinical significance until further chanical and thermal stimulation following inflammation
testing is done. However, the presence of mutations in 5% of the hindpaw. Importantly, only 11% of mice heterozy-
of this cohort suggests that a substantial number of patients gous for the mutant allele manifested self-induced tissue
with common painful neuropathies may carry mutations in lesions in the cervical area, a feature that is common to all
Nav1.9. patients reported to have this mutation. These major differ-
ences in the behavior of the transgenic mouse compared
3. Inability to experience pain with the human subjects have not been explained thus far.
Studies of three unrelated singleton individuals and one Functional testing of the L811P mutation at the channel
family identified a complex pain phenotype, including level by Leipold et al. (281) was conducted in heterologous
congenital painless bone fractures, self-mutilation, skin expression system ND7/23 DRG-derived cell line and on

BENNETT ET AL.
the endogenous Nav1.9-L799P in DRG neurons from the toms that are common to all patients with Nav1.9-related
transgenic mouse and was reported as showing a hyperpo- insensitivity to pain. The biophysical properties of the
larization of the voltage dependence of Nav1.9 channel ac- L799P channel in the transgenic mouse DRG neurons is
tivation by –28 mV, a threefold increase in current density distinctly different from the human L811P in that its inac-
and slower deactivation, all proexcitatory gain-of-function tivation is massively hyperpolarized. The divergence in the
effects. Voltage-clamp recordings in DRG neurons from the behavior of the mouse and human mutant channel should
L799P mouse showed that activation of the Nav1.9 channel not be too surprising, since these channels share only 72%
was shifted by –26 mV, comparable to the hyperpolarizing identity at the amino acid level, as discussed above. There is
shift of activation for the human mutant Nav1.9 channel in substantial evidence indicating that the reported depolar-
the ND7/23 cell line. Unlike the human Nav1.9 mutant ization of RMP of knock-in DRG neurons (281) and mouse
channel, these recordings showed a massive hyperpolariz- neurons transfected with the human L811P channel (280) is
ing shift in inactivation (–29 mV), which is expected to not likely to cause the resting inactivation of most Nav
render most of the mutant channels unavailable to open at channels in these neurons. Multiple previous studies
physiological membrane voltages. Current-clamp record- showed that depolarization of RMP by a similar magnitude
ings of DRG neurons from the L799P mouse showed a causes DRG neuron hyperexcitability (150, 151, 199, 202,
depolarizing shift of 6.7 mV in resting potential, depolar- 203, 231, 400). Indeed, the results of Leipold et al. (280)
ized voltage-threshold for action potential take-off, and a include a demonstration that a depolarization of DRG neu-
narrower action potential (281). The authors suggested that rons by 6 mV causes the hyperexcitability of DRG neurons,
the narrower action potential waveform produces less cal- although they also show a reduction in the number of these
cium influx and reduced transmitter release at presynaptic neurons that can sustain firing over an extended period, not
terminals, consistent with the reduced frequency of the likely to reflect physiological conditions.
mEPSCs that were recorded in slices from the transgenic
mouse. The authors attributed the reduced excitability of Nav1.9-related insensitivity to pain might, in principle, be
DRG neurons expressing the L799P channel to the resting explained, at least in part, by a deeper depolarization of
inactivation of sodium channels Nav1.7 and Nav1.8 in these RMP of DRG neurons that would reduce excitability via
neurons caused by the 6.7-mV depolarizing shift in resting resting inactivation of TTX-S channels and significant re-
potential. duction of the fraction of Nav1.8 channels that remain
available to open at these voltages. Indeed, functional as-
In a different set of experiments, Leipold et al. (280) trans- sessment of the L1302F and L811P in DRG neurons has
fected mouse DRG neurons with the human L811P channel now shown that the mutant channels produce a massive
and studied its effect on excitability of these neurons. Sim- depolarization of the RMP, rendering a subpopulation of
ilar to the effects of the L799P mutation in DRG neurons DRG neurons inexcitable (232). Compared with WT
from the knock-in mouse, the RMP of neurons expressing Nav1.9 channel (FIGURE 21A), the L1302F mutation (FIG-
the human L811P channel was depolarized by 5.9 mV when URE 21B) causes a large hyperpolarizing shift (⫺26.9 mV)
measured at 30°C and 5.3 mV when measured at 20°C. in the voltage dependence of activation (FIGURE 21C), but
However, other parameters of the action potential wave- no significant shift in inactivation when expressed in neu-
form were comparable or only marginally affected in neurons. The shift in activation of the L1302F mutant channels
rons transfected with WT or L811P channels. Remarkably, is comparable to that reported for the L811F mutation
evoked firing was significantly higher in neurons transfected (281). The massive shift in activation of these two mutant
with the L811P mutant channels, in contrast to the expec- channels leads to substantially enlarged window currents
tation from the pain insensitivity phenotype that the mutant (FIGURE 21D), which promote depolarization near the rest-
should render these neurons hypoexcitable. Applying a dif- ing potential of DRG neurons. It is reasonable to expect
ferent protocol of a 40-pA stimulus over a 30-s period re- that this depolarization will be much greater than that pro-
vealed a reduction in the number of neurons expressing the duced by Nav1.9 mutations that shift activation by smaller
L811P channel which maintain repetitive firing with magnitudes. In agreement with this prediction, the expres-
⬍10-mV diminution of the amplitude of the spike. The sion of L1302F and L811P in small DRG neurons evoked
authors interpreted this finding as indicating that an in- massive mean depolarizations of RMP of 11.5 mV and 8.3
creased probability of failure to maintain the firing pattern mV, respectively (232), as indicated in the superimposed
over an extended period contributes to the pain insensitivity representative action potential traces in FIGURE 22B. Even
phenotype. more informative was an examination of the range of
RMPs, which showed that a subpopulation of neurons
The mechanistic basis for the loss of sensitivity to pain in the transfected with L1302F or L811P manifested a depolar-
patients with the L811P, and presumably the L1302F, mu- ized RMP more positive than ⫺30 mV (FIGURE 22A). These
tant Nav1.9 channels in the context of the analysis above massively depolarized neurons were silent (FIGURE 22C),
has remained elusive. The L799P transgenic mouse re- but hyperpolarizing the RMP of these neurons via current
ported by Leipold et al. (281) does not recapitulate symp- injection unmasked enhanced excitability manifested as

FIGURE 21. Effects of L1302F on properties of Nav1.9 channels. Average whole cell sodium currents
recorded from ND7/23 cells stably expressing wild-type (WT; A) or L1302F (B) Nav1.9 channels are shown.
Holding potential was ⫺120 mV. The displayed current traces have been normalized to the maximum current
amplitude. Blue trace in A and gold trace in B indicate whole cell currents recorded during test depolarizations
to ⫺40 mV. C: average current-voltage relationships determined from cells expressing WT (open circles) or
L1302F (orange circles) channels. Current values were normalized to cell capacitance. Data points in C
represent means ⫾ SE. Effects of L1302F on voltage dependences of activation and inactivation are shown.
D: superimposed steady-state inactivation and activation curves calculated for WT (black) and L1302F
(orange) sodium currents. Steady-state inactivation and activation curves for WT intersect near ⫺50 mV,
whereas those for L1302F intersect at approximately ⫺70 mV. Shading indicates window current between
⫺100 and ⫺10 mV. The yellow shaded area represents the window current for L1302F channels, and the
gray area is the window current for WT channels. [Adapted with permission from Huang et al. (232).]
very-low-threshold currents (FIGURE 22D) and spontaneous microneurography data from these two patients are com-
firing. Importantly, exploration of threshold over a range of patible with increased C-fiber excitability, suggesting that
RMPs revealed a U-shaped relationship between resting po- these Nav1.9 mutations might confer gain-of-function attri-
tential and neuronal action potential threshold (FIGURE butes on the channel (260). Reduced expression of Nav1.9
23A). The reduction in action potential amplitude at pro- has been shown in some patients with Hirschsprung’s dis-
gressively depolarized membrane potential (FIGURE 23A) is ease (340).
due to inactivation of endogenous Nav1.8 channels (FIGURE
23B). This could explain why Nav1.9 mutations that evoke
small degrees of membrane depolarization cause hyperex- F. Prospects and Challenges
citability and familial episodic pain disorder or painful neu-
ropathy, whereas mutations evoking larger membrane de- The studies of Nav1.9 described in this section have confirmed
polarization cause hypoexcitability and insensitivity to an important role for this channel in human pain and support
pain. further investigations of Nav1.9 in human pain disorders. Al-
though cases of Nav1.9-related familial episodic pain and
4. Erythromelalgia and Hirschsprung disease those of Nav1.9-related insensitivity to pain are rare, they pro-
vide compelling evidence for the contribution of this channel
Nav1.9 has been linked to other pain conditions, including in human pain disorders. Studies on larger cohorts of patients
EM and Hirschsprung disease. Two mutations in Nav1.9, with pain disorders with no apparent nongenetic cause are
N1169S in DIII/S4 –5 linker and I1293V in DIII/S6, were needed to build a complete picture of Nav1.9-related pain.
recently reported in patients with a clinical phenotype re- Given the disparities between rodent models and human phe-
sembling EM and no other mutations in Nav1.7 (510). notype and channel physiology, we predict that studies of hu-
While these mutations have not been functionally tested to man channels, expressed within well-defined model systems,
date, and thus their clinical significance remains unknown, will be most informative.

BENNETT ET AL.
FIGURE 22. Effects of L1302F on resting membrane potential (RMP) and firing properties of dorsal root
ganglia (DRG) neurons. A: scatter plot of RMP recording from neurons expressing either wild-type (WT),
L811P, or L1302F Nav1.9 channel. Expression of the mutant channel L811P caused an average depolariza-
tion of RMP of 8.2 mV, whereas L1302F caused an average depolarization of 11.5 mV, compared with DRG
neurons that expressed WT channels, and this difference was statistically significant (***P ⬍ 0.001 by t-test).
Solid symbols in purple or orange indicate cells that do not fire all-or-none action potentials in response to
external stimuli at their native resting potentials, but regain excitability when held at ⫺60 mV. B: representative
recordings demonstrating that expression of L1302F (orange trace) and L811P (purple trace) is associated
with a depolarized RMP and an attenuated action potential overshoot compared with cells expressing WT
channels (blue trace). C: a small DRG neuron with an RMP of ⫺26.6 mV (indicated by arrow in A) does not fire
action potentials in response to 200-ms current injections from 0 to 500 pA in 100-pA increments. D: when
held at ⫺60 mV, the same neuron in C produces subthreshold depolarizations in responses to 30-pA and
35-pA current injections and generates action potentials with a threshold current of 40 pA. When held at ⫺60
mV, two of the other three filled orange cells fired action potentials with a threshold of 55 pA and 110 pA, and
the third one fired spontaneously. The four cells expressing L811P indicated by solid purple diamonds do not
fire action potentials in response to external stimuli at their native resting potentials but fire spontaneously
when held at ⫺60 mV (not shown here). [Adapted with permission from Huang et al. (232).]
A role for Nav1.9 in colonic-related disorders is supported by favorably to treatment with NSAIDs (280, 343, 508). A ther-
experimental data from animal studies and from patients. Pri- apeutic response to anti-inflammatory agents is consistent
mary afferents that innervate the colon of WT mice, but not with the role of Nav1.9 in inflammatory pain (15, 349, 367).
Nav1.9-null mice, increased firing when treated with an in- By contrast, patients with the R222H mutation from South
flammatory soup prepared from inflamed human gut tissue America did not respond to NSAIDs (203). Additionally, pa-
(219). A reduction in the expression of Nav1.9 in myenteric tients with Nav1.9-associated painful peripheral neuropathy
neurons, however, has been associated with Hirschsprung’s typically do not respond to NSAID treatment. This differential
disease (340). The genetic link of mutations in the RET gene to response to NSAID treatment by different individuals is not
Hirschsprung’s disease in familial and sporadic cases is strong too surprising, since these drugs do not act directly on the
(13). Functional testing of several RET mutations from pa- channel, and their signal transduction cascades and neural
tients with this disorder demonstrated loss of function (481). circuitry may be different in the nonresponsive patients.
Reduced activity of RET might be linked to reduced Nav1.9
expression, because this channel is regulated by GDNF (as The development of novel small-molecule inhibitors of
discussed earlier in this section), a trophic factor that requires sodium channels or other approaches, including gene
RET as part of its receptor. therapy and biologics that target these channels on pe-
ripheral neurons, holds promise for more effective pain
Patients with Nav1.9-related familial episodic pain R222H/S, treatments with minimal adverse effects. Patients with
R225C, A808G, and V1184A have been reported to respond early-onset Nav1.9-related insensitivity to pain and those

the development of isoform-specific blocker, and the re-

cent development of a HEK-293 stable cell line that ex-
press Nav1.9 channels (287) will undoubtedly accelerate
drug discovery. Another advantage is the preferential ex-
pression of Nav1.9 within a subset of nociceptors, which
suggests that an isoform-specific inhibitor targeting this
channel might produce fewer deficits in other sensory
modalities, and spare skeletal, cardiac, and cognitive
functions. The demonstration in rodents of Nav1.9 in
magnocellular neurosecretory neurons (47), however,
suggests that caution should be exercised in evaluating
possible side effects of inhibitors of this channel.
VI. PAIN AND NaV1.1 AND 1.6
This review has focused on the role of those VGSCs with the
strongest pedigree in pain research, namely, Nav1.3, 1.7,
1.8, and 1.9. Recent publications have also suggested a role
of two additional TTX-S VGSCs expressed by sensory neu-
rons: Nav1.1 and Nav1.6. Using two tarantula-derived al-
gogenic toxins, Osteen and colleagues (348) demonstrate
that they selectively activate Nav1.1, which elicits pain and
mechanical hypersensitivity. Selectivity for Nav1.1 over
other VGSCs is conferred by targeting two regions, S3b-S4
and S1-S2 loops in domain IV, to inhibit fast inactivation.
Most Nav1.1 expressing neurons coexpress NF200 (a
marker of myelinated neurons) or 5-HT3 (a marker of
thinly myelinated A␦ neurons) with limited overlap with
markers of unmyelinated neurons (348). These findings,
therefore, now implicate Nav1.1 expressing myelinated fi-
bers in pain processing, and this may also provide a new
molecular marker for identifying A␦ mechano-nociceptors.
FIGURE 23. Depolarization of resting membrane potential (RMP) Nav1.6 is a further TTX-S VGSC expressed by both small-
causes biphasic changes in current threshold and attenuates action and large-diameter DRG neurons (40). In myelinated sen-
potential amplitude. A: small adult dorsal root ganglia (DRG) neurons
sory afferents Nav1.6 is strongly localized at the node of
were held at membrane potentials ranging from ⫺60 mV to ⫺30
mV in 2.5-mV increments. Action potential waveforms recorded at Ranvier (62). Nav1.6 is also expressed in a continuous fash-
various voltages from representative cells are illustrated in the ion along the axon of unmyelinated C-fiber afferents and
boxed panel. The distribution of RMP in neurons from pairwise contributes to action potential conduction in these fibers
comparisons of wild-type (WT)- and L1302F-expressing neurons and (46). A number of preclinical studies have now linked
WT- and R222H-expressing neurons is illustrated by blue (WT)/
Nav1.6 to neuropathic pain states. The expression of
orange (L1302F), and green (WT)/magenta (R222H) solid circles
beneath the x-axis. Numbers in parentheses are the average RMP Nav1.6 at nodes of Ranvier increases following infra-or-
for neurons transfected with WT or mutant Nav1.9 channels. bital nerve ligation (212). After SNI, the number of de novo
***P ⬍ 0.001, *P ⬍ 0.05. Note the similar average and distribu- nodes of Ranvier increase in the neuroma, all of which
tion of the RMP of neurons transfected with WT channels, and the retain Nav1.6 expression (73). Nav1.8-Cre driven Nav1.6
marked shift toward more positive membrane potentials of RMP
knockout had no effect on acute, inflammatory or neuro-
from neurons transfected with the L1302F channels, compared
with neurons transfected with WT or the R222H channels. B: action pathic pain behaviors. However, AAV-Cre mediated
potential amplitude and RMP data were best fit by a single Boltz- Nav1.6 knockout partially attenuates SNI-induced me-
mann function with midpoint voltage of ⫺38.7 ⫾ 2.3 mV. Data chanical allodynia, suggesting Nav1.6 in Nav1.8 negative
points in A and B represent means ⫾ SE. [Adapted with permission neurons plays an important role in neuropathic pain (73).
from Huang et al. (232).]
The expression of Nav1.6 by sensory neurons was also
shown to increase in a mutant mouse model of type II dia-
with episodic pain disorder have been reported to have betes, which develops painful diabetic neuropathy (378).
normal intellectual abilities, suggesting that targeting of The chemotherapeutic agent Oxalplatin results in striking
this channel may not cause adverse cognitive deficits. The cold dysesthesia. The cold-induced hyperexcitability of sen-
relatively less conserved sequence of Nav1.9 compared sory axons induced by Oxaliplatin has been linked to en-
with the other sodium channels may be advantageous for hanced persistent and resurgent currents mediated by

BENNETT ET AL.
Nav1.6 (432). Cold allodynia in a rodent model of Oxalip- such as Carbamazepine, providing a rationale for bringing
latin-induced neuropathy was dependent on Nav1.6 (111). molecular pain medicine to the bedside.
Spontaneous activity in A␤ fibers as well as mechanical and
cold pain-related hypersensitivity were also shown to be ACKNOWLEDGMENTS
dependent on Nav1.6 in the spinal nerve ligation model of
neuropathic pain (489). Human evidence of a role for Address for reprint requests and other correspondence: D.
Nav1.6 in persistent pain has recently emerged. A novel Bennett, Nuffield Dept. of Clinical Neuroscience, John
Met136Val missense mutation in Nav1.6 was described in Radcliffe Hospital, West Wing, Level 6, Oxford OX3 9DU,
patients presenting with trigeminal neuralgia (448). This UK (e-mail: david.bennett@ndcn.ox.ac.uk).
mutation resulted in increases in peak current density with-
out altering channel gating. Nav1.6 is known to generate
GRANTS
resurgent currents in sensory neurons (101), which facili-
tate repetitive firing; the Met136Val mutation was also as-
D. L. Bennett is a Wellcome senior clinical scientist (ref. no.
sociated with increased resurgent current. Current-clamp
095698z/11/z and 202747/Z/16/Z). D. L. Bennett and A. J.
studies in TRG neurons showed that Met136Val resulted in
Clark received support from the Innovative Medicines Ini-
hyperexcitability of trigeminal neurons with a reduction in
tiative Joint Undertaking under grant agreement no.
rheobase and increased the frequency of evoked action po-
115439, resources of which are composed of financial con-
tentials in response to graded stimuli.
tribution from the European Union’s Seventh Framework
Programme (FP7/2007–2013) and European Federation of
VII. FINAL CONCLUSIONS Pharmaceutical Industries and Associations companies’ in
kind contribution. J. Huang, S. G. Waxman, and S. D.
Improved genetic sequencing technology and bioinformatic Dib-Hajj were supported by the Rehabilitation Research
analysis has revolutionized our understanding of Mende- and Development Service (B9253-C; B0395-R) and the Bio-
lian pain disorders. This has led to the identification of medical Laboratory Research and Development Service
disease-related mutations in all of the ␣-VGSC subunits (CC103), Department of Veterans Affairs, and by grants
selectively expressed within adult nociceptors (Nav1.7, 1.8, from The Erythromelalgia Association and the Taylor
and 1.9). Studying these variants has provided fundamental Foundation. The Center for Neuroscience and Regenera-
insight into both the normal function and pathophysiolog- tion Research is a Collaboration of the Paralyzed Veterans
ical role of these ion channels. This progress is likely to of America with Yale University.
continue apace and, as larger patient cohorts become avail-
able, will not only be applied to rare Mendelian pain disor- DISCLOSURES
ders but also more common acquired neuropathic pain
states, such as painful neuropathy. Such clinical progress D. L. Bennett has acted as a consultant for Abide, Glaxo
has been complemented by improved cellular and animal SmithKline, Lilly, Mundipharma, TEVA, Orion Pharma,
models to study pain biology. It is clear from these studies Pfizer, and Mitsubishi Tanabe over the last 3 yr. S. G. Wax-
that single VGSCs should not be seen in isolation, but in the man has acted as a consultant to Amgen, SiteOne Thera-
context of the integrated physiology of the nociceptive sys- peutics, Chromocell Research, Biogen, GlaxoSmithKline,
tem. This is illustrated by the important interactions be- RedPin Therapeutics, Voyager Therapeutics, and Tetrage-
tween distinct VGSCs, which can radically change the out- netics over the past 3 yr. S. D. Dib-Hajj has consulted for
come at a cellular and systems level. Guidepoint Global over the past 3 yr.
Chronic pain remains a huge unmet health need, affecting
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Physiol Rev 99: 1079 –1151, 2019

Published January 23, 2019; doi:10.1152/physrev.00052.2017

THE ROLE OF VOLTAGE-GATED SODIUM

I. INTRODUCTION 1079 complex array of ligand-gated ion channels and G protein-

0031-9333/19 Copyright © 2019 the American Physiological Society 1079

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A. Preclinical Investigations 2. Expression within the PNS: vagal sensory neurons

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B) CHANNEL REPRIMING.The firing rate of a neuron is limited by 5. Splice variants of Nav1.7

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F258fs; R830X 19304393 Compound Y Y Y Warm Normal NT Reduced NT Normal motor

R896Q 20635406 Homozygous NT Y Y NT NT Histamine normal NT NT Normal motor

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are inherited in a heterozygous fashion. This has been used

larized the membrane potential as well as increasing the

evoked by channel mutations and the clinical severity in pa-

vation are associated with a younger age of onset (95, 196).

region connects the voltage sensor (segments S1–S4) to the

properties. One such mutation in this hotspot (L858F) did not

voltage-dependent activation in a hyperpolarized direction

(FIGURE 8F). Furthermore, deactivation was markedly slower

for the L858F variant at depolarized potentials, which could

allow more channels to be open and increase the probability of

a ramp current (FIGURE 8G) (200). Studying the effect of

servation that warmth triggers pain: DRG neurons expressing

response to a warm stimulus compared with neurons express-

ing WT channels (182). Another important feature of the ef-

pressed. This was demonstrated when the L858H mutant was

expressed in DRG vs. sympathetic ganglia (199, 400). This

activation of Nav1.8 (which has a more depolarized voltage

dependence of activation than Nav1.7), leading to hyperexcit-

ability. Sympathetic neurons, however, which do not express

mutation based on depolarization-induced inactivation of

Pain associated with IEM can be extremely difficult to treat,

treatment. Drugs such as anticonvulsants and local anes-

thetics, which have broad activity against VGSCs, are often

physiological level) is dependent on the exact mutation.

Carbamazepine and mexiletine have shown efficacy in the

context of certain mutations (82, 94, 495). A recent ad-

vance has been the use of structural modeling to predict

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Q10R 19369487 29A⬎G Hyperpol UC UC Enhanced Slower NT UC No NT UC Reduced Increased

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Carbamazepine is an effective treatment at reducing the