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Molecular Physiology of Cardiac Repolarization

JEANNE M. NERBONNE AND ROBERT S. KASS


Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis,
Missouri; and Department of Pharmacology, College of Physicians and Surgeons,
Columbia University, New York, New York
I. Introduction 1206
II. Myocardial Action Potentials and Voltage-Gated Inward Sodium and Calcium Currents 1208
A. Voltage-gated Na

(Nav) currents 1208


B. Voltage-gated Ca
2
(Cav) currents 1209
III. Myocardial Action Potentials and Repolarizing Voltage-Gated Potassium Currents 1211
A. Transient outward Kv currents 1211
B. Delayed rectifier Kv currents 1213
C. Regional differences in Kv current expression and properties 1214
IV. Other Myocardial Potassium Currents Contributing to Repolarization 1214
V. Molecular Components of Myocardial Nav and Cav Channels 1216
A. Nav channel pore-forming -subunits 1216
B. Nav channel accessory subunits and other interacting proteins 1219
C. Cav channel pore-forming -subunits 1222
D. Cav channel accessory subunits and other interacting proteins 1223
VI. Molecular Components of Myocardial Kv Channels 1225
A. Kv channel pore-forming -subunits 1225
B. Kv channel accessory subunits 1227
C. Molecular correlates of cardiac transient outward Kv channels 1230
D. Molecular correlates of cardiac delayed rectifier Kv channels 1232
VII. Molecular Components of Other Cardiac Potassium Channels 1234
A. Inwardly rectifying cardiac K

(Kir) channel pore-forming -subunits 1234


B. Two-pore domain K

(K2P) channel pore-forming -subunits 1236


VIII. Myocardial Potassium Channels and the Actin Cytoskeleton 1237
IX. Summary and Conclusions 1238
Nerbonne, Jeanne M., and Robert S. Kass. Molecular Physiology of Cardiac Repolarization. Physiol Rev 85:
12051253, 2005; doi:10.1152/physrev.00002.2005.The heart is a rhythmic electromechanical pump, the functioning of
which depends on action potential generation and propagation, followed by relaxation and a period of refractoriness until
the next impulse is generated. Myocardial action potentials reect the sequential activation and inactivation of inward
(Na

and Ca
2
) and outward (K

) current carrying ion channels. In different regions of the heart, action potential
waveforms are distinct, owing to differences in Na

, Ca
2
, and K

channel expression, and these differences contribute


to the normal, unidirectional propagation of activity and to the generation of normal cardiac rhythms. Changes in channel
functioning, resulting from inherited or acquired disease, affect action potential repolarization and can lead to the
generation of life-threatening arrhythmias. There is, therefore, considerable interest in understanding the mechanisms
that control cardiac repolarization and rhythmgeneration. Electrophysiological studies have detailed the properties of the
Na

, Ca
2
, and K

currents that generate cardiac action potentials, and molecular cloning has revealed a large number
of pore forming () and accessory (, , and ) subunits thought to contribute to the formation of these channels.
Considerable progress has been made in dening the functional roles of the various channels and in identifying the
-subunits encoding these channels. Much less is known, however, about the functioning of channel accessory subunits
and/or posttranslational processing of the channel proteins. It has also become clear that cardiac ion channels function
as components of macromolecular complexes, comprising the -subunits, one or more accessory subunit, and a variety
of other regulatory proteins. In addition, these macromolecular channel protein complexes appear to interact with the
actin cytoskeleton and/or the extracellular matrix, suggesting important functional links between channel complexes, as
well as between cardiac structure and electrical functioning. Important areas of future research will be the identication
of (all of) the molecular components of functional cardiac ion channels and delineation of the molecular mechanisms
involved in regulating the expression and the functioning of these channels in the normal and the diseased myocardium.
Physiol Rev 85: 12051253, 2005;
doi:10.1152/physrev.00002.2005.
www.prv.org 1205 0031-9333/05 $18.00 Copyright 2005 the American Physiological Society
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I. INTRODUCTION
The normal mechanical (pump) functioning of the
mammalian heart depends on proper electrical function-
ing (56, 173), reected in the sequential activation of cells
in specialized, pacemaker regions of the heart and the
propagation of activity through the ventricles (Fig. 1).
Myocardial electrical activity is attributed to the genera-
tion of action potentials in individual cardiac cells, and
the normal coordinated electrical functioning of the
whole heart is readily detected in surface electrocardio-
grams (Fig. 1). The propagation of activity and the coor-
dination of the electromechanical functioning of the ven-
tricles also depend on electrical coupling between cells,
mediated by gap junctions (251, 435). The generation of
myocardial action potentials reects the sequential acti-
vation and inactivation of ion channels that conduct de-
polarizing, inward (Na

and Ca
2
), and repolarizing, out-
ward (K

), currents (24, 375). The waveforms of action


potentials in different regions of the heart are distinct
(Fig. 1), owing to differences in the expression and/or the
properties of the underlying ion channels (24, 374). These
differences contribute to the normal unidirectional prop-
agation of excitation through the myocardium and to the
generation of normal cardiac rhythms (23, 24, 259, 374,
375). Changes in the properties or the functional expres-
sion of myocardial ion channels, resulting from inherited
mutations in the genes encoding these channels (23, 36,
51, 102, 204, 243, 253) or from myocardial disease (34, 49,
67, 184, 365, 496, 501503, 510), can lead to changes in
action potential waveforms, synchronization, and/or
propagation, thereby predisposing the heart to potentially
life-threatening arrhythmias (13, 14, 16, 24, 127, 259, 436).
There is, therefore, considerable interest in delineating
the molecular, cellular, and systemic mechanisms con-
tributing to the generation and maintenance of normal
cardiac rhythms, as well as in understanding how these
mechanisms are altered in the diseased myocardium.
Myocardial electrical activity is initiated in the pace-
maker cells in the sinoatrial (SA) node and then propa-
gated through the atria to the atrioventricular (AV) node
(Fig. 1). Following a brief pause in the AV node, excitation
spreads in the conducting Purkinje bers to the apex of
the heart and into the working, ventricular myocardium
(Fig. 1). In cells in each of these specialized regions,
excitation results in action potential generation, followed
by relaxation and a period of refractoriness until the next
impulse is generated and propagated. The observed het-
erogeneity in action potential waveforms in different cell
types (Fig. 1) reects differences in ion channel expres-
sion levels, and modeling studies suggest that small
changes in the time- and/or voltage-dependent properties
of cardiac sarcolemmal ion channels can have rather
profound effects on action potential durations, as well as
impact refractoriness and rhythmicity (105, 127, 311, 312).
In ventricular and atrial myocytes and in Purkinje bers
(Fig. 1), the upstroke of the action potential (phase 0) is
rapid, resulting from the activation of voltage-gated Na

FIG. 1. Electrical activity in the myo-


cardium. Top: schematic of a human
heart with illustration of typical action
potential waveforms recorded in differ-
ent regions. Bottom: schematic of a sur-
face electrocardiogram; three sequential
beats are displayed.
1206 JEANNE M. NERBONNE AND ROBERT S. KASS
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(Nav) channels (172) (Fig. 2). In pacemaker cells in the SA
node (SAN) and AV node (AVN), however, phase 0 is
markedly slower than in atria/ventricles (Fig. 1), suggest-
ing that Nav channels do not play a prominent role in
depolarization. Nevertheless, Nav currents have been de-
scribed in subsets of rabbit and guinea pig AVN cells (361,
579), as well as in rabbit SAN cells (173). The properties
of the Nav currents expressed in cardiac cells from dif-
ferent species, as well as in different cell types in the
same species (Table 1), are similar, an observation that
might be interpreted as suggesting that the molecular
correlates of the underlying channels are the same (see
sect. VA).
Phase 0 of the action potential in Purkinje bers and
in atrial and ventricular myocytes is followed by a tran-
sient repolarization (phase 1), reecting Nav channel in-
activation and the activation of the fast transient voltage-
gated outward K

current (I
to,f
) (Fig. 2). This transient
repolarization or notch, which can be quite prominent in
Purkinje and ventricular cells (Fig. 1), inuences the
height and duration of the action potential plateau (phase
2). Membrane depolarization also activates voltage-gated
Ca
2
(Cav) currents, and the inux of Ca
2
through L-
type Cav channels during the phase 2 plateau is the main
trigger for excitation-contraction coupling in the working
myocardium (57, 154). In SAN and AVN cells, activation of
(L-type) Cav channels also contributes to action potential
generation, particularly in cells expressing low levels of
functional Nav channels (57). In some cardiac cells/spe-
cies, another class of Cav channels, the T-type Cav chan-
nels (Table 1), has been distinguished and suggested to
play a role in automaticity (57, 394). As with cardiac Nav
channels, however, the properties of the L- and T-type
cardiac Cav channels characterized electrophysiologi-
cally in different cell types and in different species (Table
1) are quite similar, suggesting that the molecular corre-
lates of the underlying L- and T-type (Cav) channels are
also similar throughout the myocardium (see sect. VC).
The driving force for K

efux is high during the


plateau phase of the action potential in ventricular and
atrial myocardium and, as the Cav channels inactivate, the
outward K

currents predominate, resulting in (phase 3)


repolarization, bringing the membrane voltage back to the
resting potential (Fig. 2). In contrast to Nav and Cav
currents, however, there are multiple types of voltage-
gated K

(Kv) currents, as well as non-voltage-gated,


inwardly rectifying K

(Kir) currents (Table 1), that con-


tribute to myocardial action potential repolarization. The
greatest functional diversity is among Kv channels (Table
1). At least two types of transient outward currents, I
to,f
and I
to,s
, and several components of delayed rectication,
including I
Kr
[I
K(rapid)
], I
Ks
[I
K(slow)
], and I
Kur
[I
K(ultrarapid)
],
for example, have been distinguished (Table 1). The time-
and voltage-dependent properties of the various Kv cur-
rents identied in myocytes isolated from different spe-
cies and/or from different regions of the heart in the same
species, however, are remarkably similar, suggesting that
the same (or very similar) molecular entities contribute to
the generation of each of the various types of Kv channels
(Table 1) in different cells/species. The relative Kv chan-
nel expression levels vary in cardiac cells in different
regions (i.e., atria, ventricles) of the heart, and this hetero-
FIG. 2. Action potential waveforms and
underlying ionic currents in adult human and
ventricular (left) and atrial (right) myocytes.
The time- and voltage-dependent properties
of the voltage-gated inward Na

(Nav) and
Ca
2
(Cav) currents expressed in human
atrial and ventricular myocytes are similar.
In contrast, there are multiple types of K

currents, particularly Kv currents, contribut-


ing to atrial and ventricular action potential
repolarization. The properties of the various
Kv currents are distinct, and in contrast to
the inward currents, there are multiple Kv
currents expressed in individual myocytes
throughout the myocardium.
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geneity contributes importantly to the observed regional
differences in action potential waveforms (24, 127, 373,
374). Changes in the properties or the functional expres-
sion of Kv channels, as occurs in a variety of myocardial
diseases (34, 49, 67, 365, 496, 503, 510), can, therefore,
have dramatic effects on action potential waveforms,
propagation, and rhythmicity.
A large number of pore-forming () subunits, encod-
ing Nav, Cav, Kv, and Kir channels, and a variety of
channel accessory (, , and ) subunits have been iden-
tied (Tables 26), and considerable progress has been
made in dening the expression patterns of these subunits
in the heart and the roles of the individual subunits in the
generation of functional cardiac (Nav, Cav, Kv, and Kir)
channels (Tables 26). These studies have demonstrated
that distinct molecular entities underlie the various car-
diac ion channels/currents that have been distinguished
electrophysiologically and shown to contribute to myo-
cardial action potential repolarization. It also has now
been shown that mutations in the genes encoding the
subunits involved in the generation of functional cardiac
Nav, Kv, Cav, and Kir channels underlie several inherited
cardiac arrhythmias (23, 36, 51, 102, 204, 253, 479, 481).
Although inherited rhythm disorders are rare, these mu-
tations belong to an ever-increasing number of chan-
nelopathies, i.e., diseases linked to genes encoding ion
channels (35, 123, 204, 220, 234, 243, 267, 309, 359, 403,
442, 459). Based on the rapid progression of this eld
(249) and the growing molecular complexity of ion chan-
nels, it seems certain that the number of genes encoding
ion channels or ion channel regulatory molecules linked
to inherited and acquired disorders of the cardiovascular
(and other) system will continue to increase, perhaps
dramatically, in the future. The densities and the func-
tional properties of myocardial Nav, Cav, Kv, and Kir
currents also change in a number of acquired myocardial
disease states (34, 49, 67, 365, 496, 503, 510), and these
changes can lead to the generation of potentially life-
threatening cardiac arrhythmias. At present, therefore,
there is considerable interest in understanding the de-
tailed molecular mechanisms controlling the properties
and the functional cell surface expression of the various
ion channels controlling myocardial action potential re-
polarization, as well as the impact of genetic and epige-
netic factors, including cardiac and noncardiac disease,
on the functioning of these channels.
II. MYOCARDIAL ACTION POTENTIALS AND
VOLTAGE-GATED INWARD SODIUM AND
CALCIUM CURRENTS
A. Voltage-Gated Na

(Nav) Currents
Voltage-gated cardiac Na

(Nav) channels open rap-


idly on membrane depolarization (Fig. 2) and underlie the
rapidly rising phases of the action potentials recorded in
mammalian ventricular and atrial myocytes and in cardiac
Purkinje bers (93, 375). Although not evident in all cells,
Nav channels with similar properties are also expressed
in subsets of mammalian SAN and AVN cells, and differ-
ences in functional Nav channel expression likely contrib-
TABLE 1. Cardiac currents contributing to action potential repolarization
Channel Type Current Name Activation Inactivation Recovery Species Tissue
Nav
I
Na
Very fast Fast Fast Cat, dog, ferret, human, mouse, rat A, P, V, SAN, AVN
Cav
I
Ca(L)
Fast Moderate* Fast Cat, dog, ferret, human, mouse, rat A, P, V, SAN, AVN
I
Ca(T)
Fast Fast Slow Cat, dog, guinea pig, rat A, P, SAN, AVN
Kv (I
to
)
I
to, t
Fast Fast Fast Cat, dog, ferret, human, mouse, rat A, P, V
I
to,s
Fast Moderate Slow Ferret, human, mouse, rat, rabbit V (A, AVN, SAN)
Kv (I
k
)
I
Kr
Moderate Fast Slow Cat, dog, guinea pig, human, mouse, rabbit, rat A, P, V, SAN, AVN
I
Ks
Very slow No Dog, guinea pig, human, rabbit A, P, V, SAN
I
Kur
Very fast Very slow Slow Dog, human A
I
K,slow1
Very fast Slow Slow Mouse A, V
I
Kp
Fast No Guinea pig V
I
K,slow2
Fast Very slow Slow Mouse A, V
I
K
Slow Slow Slow Rat V
I
ss
Slow No Dog, human, mouse, rabbit, rat A, V, AVN
Kir
I
Kl
Cat, dog, ferret, human, mouse, rabbit, rat A, P, V
A, atrial; P, Purkinje; V, ventricular; SAN, sinoatrial node; AVN, atrioventricular node. The dashed boxes are placed around currents given
different names in different species that likely are encoded by the same channels subunit genes. * Inactivation is Ca
2
and voltage dependent.
Observed in some, but not all, AVN and SAN cells. Only in nonventricular cells in rabbit.
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ute to action potential heterogeneity in pacemaker cells
(262, 361, 579, 582). Although the properties of the Nav
channels expressed in different cardiac cells are similar,
the biophysical and pharmacological properties of these
channels are distinct from Nav channels expressed in
other excitable cells, such as neurons and skeletal muscle
(93, 573). Cardiac Nav channels, for example, are remark-
ably insensitive to the Nav channel toxin tetrodotoxin
(TTX), which binds with high (nM) afnity to neuronal
and skeletal muscle Nav channels and blocks Na

inux
(93, 573). This observation was probably the rst indica-
tion that the molecular identities of the Nav channels in
cardiac myocytes, neurons, and skeletal muscle were dis-
tinct, and as detailed in section VA, this has now been
demonstrated.
On membrane depolarization, cardiac Nav channels
activate and inactivate rapidly (172, 174). The threshold
for Nav channel activation is quite negative (approxi-
mately 55 mV), and the activation of these channels is
steeply voltage dependent. Importantly, inactivation is
also voltage dependent, and cardiac Nav channels can
undergo voltage-dependent inactivation without ever
opening (174). Nevertheless, persistent openings of car-
diac Nav channels are occasionally observed, even at
depolarized membrane potentials (437, 591). At potentials
corresponding to the action potential plateau in ventric-
ular myocytes, present estimates are that 99% of the Nav
channels are in an inactivated, nonconducting state (423,
525). There is, therefore, a nite, albeit small (1%),
probability of Nav channels being open at potentials cor-
responding to the action potential plateau (525). A slow
component of Nav channel inactivation has indeed been
described in normal human ventricular myocytes (323).
This current is modulated by lysolipids (501) and appears
to be upregulated in failing myocardium (501503). Im-
portantly, the inward (Na

) current through open Nav


channels during the action potential plateau (phase 2) will
counter the effects of the increased K

efux, thereby
slowing or delaying repolarization and increasing action
potential durations (23, 102). It follows, therefore, that
changes in Nav channel open probability at voltages cor-
responding to the plateau (i.e., phase 2) could markedly
affect action potential waveforms, particularly in ventric-
ular cells.
The probability of Nav channel opening at depolar-
ized potentials (i.e., during phase 2) is determined by the
overlap of the curves describing the voltage dependences
of channel activation and inactivation (30). At the molec-
ular level, the fact that some channels are open over this
voltage window implies that there is a nite probability
that inactivation is reversible, i.e., that inactivated chan-
nels can reopen at depolarized potentials. Consistent with
these predictions, electrophysiological studies reveal the
presence of a sustained component of inward Nav cur-
rent, i.e., a persistent Nav current, during prolonged
membrane depolarizations. Although the persistent Nav
channel (window) current is small, this current could, in
principle, contribute to determining action potential du-
rations (438, 439). In this context, it is interesting to note
that it has been reported that the expression level of the
persistent Nav current component varies in different re-
gions of the ventricles (438), differences that could con-
tribute to the observed regional heterogeneities in ven-
tricular action potential durations (24, 374). The concept
that small inward (Na

) currents could profoundly affect


action potential waveforms and excitability in the myo-
cardium was suggested more than 50 years ago in the
pioneering studies of Silvio Weidman (542). The impact of
alterations in the persistent Nav channel window cur-
rent on cardiac rhythms has now been denitively dem-
onstrated with the identication of mutations in the gene,
SCN5A, encoding the TTX-insensitive cardiac Nav chan-
nels (see sect. VA) in patients with an inherited form of
long QT syndrome, LQT3 (52). A number of SCN5A mu-
tations in different affected individuals/families have been
identied (Fig. 3A) and linked to Brugada syndrome and
to conduction defects, in addition to LQT3 (23, 36, 51, 60,
93, 102, 105, 204, 253, 422, 519, 525, 540). Interestingly,
mutations in noncardiac Nav channel genes have also
been linked to familial paroxysmal dysfunction in the
skeletal and nervous systems (123, 204, 220, 359).
B. Voltage-Gated Ca
2
(Cav) Currents
In contrast to skeletal muscle, it has long been rec-
ognized that Ca
2
entry from the extracellular space is
required for excitation-contraction coupling in the mam-
malian myocardium (56, 57, 154, 173). The pathway for
plasmalemmal Ca
2
entry was rst revealed in voltage-
clamp recordings from multicellular (frog) atrial prepara-
tions and was termed the slow inward current pathway
(416418, 429). Subsequent studies revealed that this
slow inward current is carried by Ca
2
through a mem-
brane conductance distinct from the voltage-dependent
(Nav channel) pathway for Na

movement (45, 332, 386,


416418, 429). Further studies detailed the time- and
voltage-dependent properties of voltage-gated cardiac
Ca
2
(Cav) currents, rst, in multicellular preparations
and later, in isolated single cardiac cells (45, 57, 172,
416418).
Although the presence of two functionally distinct
types of Cav currents in single (starsh egg) cells was rst
reported in 1975 (201), it was not until the late 1980s that
the import and the generality of these observations be-
came clear. Two types of Cav currents/channels, for ex-
ample, were clearly distinguished in (chick and rat) sen-
sory neurons, based primarily on differences in the
thresholds for channel activation (85, 86). These channels
were termed high voltage-activated (HVA) and low volt-
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age-activated (LVA) Cav channels. Cardiac HVA and LVA
Cav channels were rst described in isolated canine atrial
cells (44). LVA Cav channels, also referred to as T-type
Ca
2
channels (394), activate at relatively hyperpolarized
membrane potentials, i.e., approximately 50 mV, and
these channels activate and inactivate rapidly (85, 86,
382). HVA Cav channels, in contrast, open on depolariza-
tion to membrane potentials positive to approximately
20 mV, and these channels inactivate over a time course
of several tens of milliseconds to seconds, depending on
the preparation and the charge carrying ion (85, 326).
Under physiological conditions, with Ca
2
as the charge
carrier, HVA channels in most cells inactivate in 100 ms
at depolarized voltages (44, 326).
The detailed kinetic, pharmacological, and voltage-
dependent properties of HVA Cav channels in different
cell types are distinct, suggesting that HVA Cav channels
are heterogeneous, particularly compared with LVA Cav
channels. Consistent with this view, multiple types of
HVA channels have now been identied in different cell
types, and these are referred to as L, N, P, Q, or R
channels (276, 382, 394). Although all HVA Cav channels
exhibit relatively large single-channel conductances
(1325 pS) and have similar permeation properties, the
detailed biophysical properties and the pharmacological
sensitivities of the various types of HVA Cav channels are
distinct. In the mammalian heart, L-type HVA Cav cur-
rents appear to be ubiquitously expressed (44, 49, 326). In
addition, the properties and the densities of L-type Cav
channel currents in cells isolated from different regions of
the myocardium, as well as in cardiac cells from different
species, are quite similar, suggesting that the molecular
compositions of the underlying channels and the molec-
ular mechanisms controlling the functional expression of
these channels are the same. Importantly, however, the
time- and voltage-dependent properties of cardiac L-type
HVA currents are distinct from the L-type HVA Cav cur-
rents expressed in skeletal muscle and in neurons (44,
326, 340), suggesting that, similar to the Nav channels,
distinct molecular entities underlie the L-type HVA Cav
channels in different tissues (see sect. VC).
The opening of cardiac L-type Cav channels in re-
sponse to membrane depolarization is delayed relative to
the Nav channels (Fig. 2), and these channels, therefore,
contribute little to phase 0 depolarization in Purkinje,
atrial and ventricular cells. Rather, the opening of HVA
L-type Cav channels and the Ca
2
entry through these
channels underlies the action potential plateau (phase 2),
which is particularly prominent in ventricular and Pur-
kinje cells (Fig. 2). In addition, Ca
2
inux through the
L-type HVA Cav channels triggers Ca
2
release from in-
tracellular Ca
2
stores and underlies excitation-contrac-
tion coupling in the working (ventricular) myocardium
(56, 57, 154, 173). L-type HVA Cav channels are also
expressed in SAN and AVN cells, where they play a role in
action potential generation, as well as in regulating auto-
maticity (49, 72, 262, 340, 361, 579). Cardiac L-type HVA
Cav channels undergo rapid voltage- and Ca
2
-dependent
inactivation (166, 281, 326), processes that will also inu-
ence action potential waveforms (Fig. 2) by affecting the
duration of the plateau (phase 2) and the time course of
action potential repolarization.
FIG. 3. Pore-forming () subunits of
cardiac Nav (A) and Kv (B and C) chan-
nels linked to inherited arrhythmias. A:
membrane topology of the four domains
(IIV) of SCN5A is illustrated with muta-
tions linked to LQT3 and Brugada syn-
dromes and to conduction disorders.
Schematics illustrating the transmem-
brane topologies of KCNH
2
(B) and
KCNQ1 (C) subunits with the mutations
linked to LQT2 and LQT1, respectively,
depicted by the open and closed circles.
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In addition to the ubiquitously expressed HVA L-type
cardiac Cav currents, LVA or T-type Cav channel currents
have also been identied in voltage-clamp recordings
from adult atrial myocytes and conducting tissues in sev-
eral different species (44, 200, 340, 394). Although not
evident in normal adult ventricular myocytes (394), T type
Cav currents have also been recorded in neonatal rat and
rabbit ventricular myocytes (547, 548). In addition, it has
been demonstrated that T-type Cav currents are ex-
pressed in ventricular myocytes in several animal models
of ventricular hypertrophy (328, 383), ndings consistent
with the view that substantial remodeling occurs in the
hypertrophied myocardium, reecting a reversion to a
fetal/neonatal pattern of gene expression (486). It is cer-
tainly possible that similar remodeling occurs in the hy-
pertrophied human heart (49). Nevertheless, it is impor-
tant to note that, to date, T-type LVA Cav channels have
not been detected in normal or diseased human myocar-
dial cells (49, 394).
In addition to marked differences in biophysical
properties, the physiological role(s) of cardiac T-type LVA
Cav channels appears to be quite different from the L-type
HVA Cav channels. As noted previously, for example,
Ca
2
entry through L-type channels in cardiac cells re-
sults in Ca
2
-induced Ca
2
release from intracellular
(Ca
2
) stores and is the main trigger for excitation-con-
traction coupling (57, 154). Although Ca
2
entry through
T-type channels also triggers Ca
2
release from intracel-
lular stores, the coupling is less efcient (589), and it
seems unlikely that T channels contribute importantly to
excitation-contraction coupling. This may simply reect
the fact that LVA channel densities are low and that,
owing to rapid inactivation, very little Ca
2
actually en-
ters cells on depolarization. Alternatively, these func-
tional differences may reect the fact that HVA and LVA
cardiac Cav channels are differentially localized, i.e., L-
type, but not T-type, Cav channels are highly localized in
the t tubules near the storage sites for intracellular Ca
2
sequestration/release (394, 589). Further experiments will
be necessary to determine the mechanistic basis for the
distinct functional roles of L- and T-type Ca
2
channels in
regulating excitation-contraction coupling.
The nding that T-type LVA Cav currents activate at
relatively hyperpolarized potentials and that these chan-
nels are expressed preferentially in pacemaker and con-
ducting cells in the heart suggests the interesting possi-
bility that there is a role for LVA channels in pacemaking
(200). Although some experimental support for this hy-
pothesis has been provided, rigorous testing is compli-
cated by the paucity of highly selective LVA Cav channel
blockers (394). In addition, it has been reported that
(rabbit) SAN cells express rapidly activating Na

-depen-
dent inward currents that are blocked by Ca
2
channel
blockers (582). These observations suggest that pace-
maker cells may express additional novel inward currents
that contribute to shaping action potential waveforms and
to regulating normal cardiac rhythms. Additional studies,
focused on further characterization of the properties of
LVA Cav channels in SAN and AVN cells and determina-
tion of the functional roles of these channels in regulating
pacemaking, will be needed to explore these possibilities
directly.
III. MYOCARDIAL ACTION POTENTIALS AND
REPOLARIZING VOLTAGE-GATED
POTASSIUM CURRENTS
Voltage-gated K

(Kv) channels are the primary de-


terminants of action potential repolarization in the mam-
malian myocardium, and compared with cardiac Nav and
Cav channels, there is considerable electrophysiological
and functional cardiac Kv channel diversity (42, 373, 375).
Based primarily on differences in time- and voltage-de-
pendent properties and pharmacological sensitivities (42,
373, 375), two broad classes of repolarizing cardiac Kv
currents have been distinguished (42): transient outward
K

currents (I
to
) and delayed, outwardly rectifying K

currents (I
K
) (Table 1). The transient currents (I
to
) acti-
vate and inactivate rapidly on membrane depolarizations
to potentials positive to approximately 30 mV and un-
derlie the early phase (phase 1) of repolarization in ven-
tricular and atrial cells (Fig. 2). Cardiac delayed rectiers
(I
K
) activate at similar membrane potentials and with
variable kinetics, and these currents determine the latter
phase (phase 3) of repolarization back to the diastolic
potential (Fig. 2). Multiple types of myocardial I
to
and I
K
channels with distinct time- and voltage-dependent prop-
erties (Table 1), however, have been identied, and dif-
ferences in the densities and the biophysical properties of
these channels contribute to variations in the waveforms
of action potentials recorded in different cardiac cell
types (Fig. 1), as well as in different species (24, 374, 375,
436). The detailed pharmacological, time- and voltage-
dependent properties of each of the various repolarizing
Kv currents characterized in different cardiac cell types
and species are quite similar, thereby allowing Kv chan-
nels to be classied based on these biophysical properties
(Table 1). The observed similarities in Kv channel prop-
erties also suggest that the molecular correlates of the
underlying Kv channels are also similar (42, 373), and
considerable experimental evidence has now been pro-
vided to support this hypothesis (see sect. VI).
A. Transient Outward Kv Currents
Although cardiac transient outward currents were
rst described in (sheep) Purkinje bers and thought to
reect Cl

conductances (143, 175), subsequent work


demonstrated the presence of two transient outward cur-
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rents with distinct properties and referred to as I
to1
and
I
to2
(112). Pharmacological studies revealed that I
to1
is
blocked by 4-aminopyridine (4-AP) and unaffected by
changes in extracellular Ca
2
, whereas I
to2
is not blocked
by 4-AP and is Ca
2
dependent (257, 256). In further
studies, it was shown that the Ca
2
-dependent I
to2
in
Purkinje bers and ventricular cells is a Cl

(not a K

)
current (593, 594). In contrast, the Ca
2
-independent
component, I
to1
, was shown to be K

selective (594), and


transient outward K

currents, referred to variably by


different laboratories as I
to
, I
to1
, or I
t
(42, 83, 436), have
now been described in many cardiac cell types and in
most species. Comparison of the detailed biophysical
properties of the transient outward K

currents described
in various cell types/species, however, suggested there
might actually be two types of transient outward K

currents (42), and electrophysiological and pharmacolog-


ical studies have now provided considerable support for
this hypothesis. In adult mouse ventricular myocytes, for
example, two transient K

currents, termed I
to,fast
(I
to,f
)
and I
to,slow
(I
to,s
), have been distinguished (562). On mem-
brane depolarization, mouse ventricular I
to,f
channels ac-
tivate and inactivate rapidly, and on membrane repolar-
ization, these (I
to,f
) channels recover rapidly from steady-
state inactivation (562). In the adult mouse, I
to,f
channels
contribute importantly to the rapid repolarization of ac-
tion potentials (194, 562) that is likely necessary to main-
tain the very high resting heart rates (700 beats/min) in
these animals. In humans and other larger mammals, I
to,f
underlies the early phase (phase 1) of repolarization in
ventricular and atrial cells (Fig. 2) and likely also contrib-
utes to determining the plateau (phase 2).
Similar to I
to,f
, mouse ventricular I
to,s
channels acti-
vate and inactivate rapidly (562). In contrast to I
to,f
, how-
ever, I
to,s
channels recover very slowly (time constants of
seconds) from (steady-state) inactivation and are func-
tionally distinct from I
to,f
channels (196, 562). In addition,
I
to,f
is readily distinguished from other Kv currents, in-
cluding I
to,s,
(562), using the spider K

channel toxins
Heteropoda toxin-2 or -3 (449). The distinct properties of
I
to,f
and I
to,s
suggested that these currents reect the
functioning of two molecularly distinct Kv channels, and
considerable evidence has now been provided to support
this hypothesis (see sect. VIC). Detailed comparisons of
the properties of the transient outward K

currents ex-
pressed in other species, whether termed I
to
, I
to1
, or I
t
,
suggest that, in each case, these currents could also be
classied as I
to,s
or I
to,f
, based on the kinetics of current
inactivation and recovery from steady-state inactivation,
as well as by the differential sensitivities of the channels
to the Heteropoda toxins. Although the properties of the
transient K

currents in different cell types and species


are similar and are amenable to classication as either I
to,f
or I
to,s
(Table 1), there are differences in the detailed
biophysical properties of the (I
to,f
and I
to,s
) currents in
different cells/species (15). These observations suggest
that there may well be subtle, albeit potentially important,
molecular heterogeneity among I
to,f
and I
to,s
channels in
different cell types and/or in different species (see
sect. VIC).
Although originally identied in Purkinje bers, I
to,f
is a prominent repolarizing current in atrial and ventric-
ular myocytes in most species (26, 58, 65, 69, 75, 79, 160,
178, 264, 294, 297, 500, 530, 545, 546, 578), including
humans. Nevertheless, there are exceptions. In guinea pig
ventricular cells, for example, I
to,f
has not been detected
except when extracellular Ca
2
is removed (224). In ad-
dition, I
to,f
is not detected in rabbit atrial or ventricular
cells (156, 160, 183, 530). Nevertheless, there are transient
Kv currents in rabbit myocytes (typically referred to as I
t
),
which inactivate slowly and recover from (steady-state)
inactivation very slowly (160, 530). The properties of
these currents, therefore, more closely resemble mouse
ventricular I
to,s
than I
to,f
(562). Similar to the mouse,
however, two distinct transient outward Kv currents have
been described in the ventricles of other mammals (77,
178, 294, 500), including humans (225, 264, 545, 546), and
the properties of these currents are quite similar to those
of mouse ventricular I
to,f
and I
to,s
(79, 196, 562), permit-
ting their classication as such (Table 1). In ferret, the
rates of inactivation and recovery (from steady-state in-
activation) of the transient Kv currents in myocytes iso-
lated from the left ventricular (LV) endocardium are sig-
nicantly slower than the currents in cells from the epi-
cardial surface of the LV, suggesting the presence of two
distinct transient Kv currents that are differentially ex-
pressed (77). Examination of the reported biophysical
properties (77) suggests that the endocardial and epicar-
dial LV currents can be classied as I
to,s
and I
to,f
, respec-
tively (Table 1). The distinct transient Kv currents in the
epicardial, midmyocardial, and endocardial layers of ca-
nine (294, 500) and human (264, 545, 546) ventricles can
also be appropriately referred to as I
to,f
or I
to,s
(Table 1).
Transient Kv currents that can be classied as I
to,f
(Table 1) have also been shown to be expressed in (rab-
bit) SAN cells, although, similar to Nav currents, I
to,f
densities vary markedly among (SAN) cells (213, 283). I
to,f
densities are higher, for example, in the larger cells iso-
lated from the periphery, compared with the smaller cells
in the center, of the SAN (213, 283). In addition, when
expressed, I
to,f
appears to play a role in shaping action
potential waveforms and in regulating automaticity in
SAN cells (72, 213, 283). Cells isolated from the (rabbit)
AVN also express I
to,f
(349, 361, 371), and detailed kinetic
analysis of the currents reveals the presence of two com-
ponents with distinct rates of inactivation and recovery
(349). It is unclear whether these ndings reect differ-
ences in the kinetic properties of a single type of I
to,f
channel or if two distinct types of I
to
channels are ex-
pressed in (rabbit) AVN cells. Similar to the (rabbit) SAN,
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there is considerable heterogeneity in I
to
densities among
(rabbit) AVN cells (349). In contrast to SAN cells (213,
283), however, the differences in I
to,f
densities are not
correlated with cell size in AVN cells (349). Interestingly,
and similar to ndings in guinea pig atrial and ventricular
cells, I
to,f
is not detected in guinea pig AVN cells (579). It
is presently unclear, however, whether currents with
properties similar to ventricular I
to,s
are expressed in
conducting tissues in guinea pig heart. Owing to the
marked differences in inactivation and recovery kinetics
of I
to,f
and I
to,s
channels, however, the differential expres-
sion of these two channel types would be expected to
have profound functional effects on the regulation of
rhythmicity in the normal heart, effects that will be aug-
mented in the diseased myocardium.
B. Delayed Rectier Kv Currents
Myocardial delayed rectier Kv currents, I
K
, also rst
described in (sheep) Purkinje bers (379), have been
characterized in atrial and ventricular myocytes, as well
as in pacemaker cells, isolated from a variety of different
species, and, in most cases, multiple components of I
K
are
coexpressed (Table 1). In guinea pig ventricular and atrial
myocytes, for example, two prominent components of I
K
,
I
Kr
(I
K,rapid
) and I
Ks
(I
K,slow
), were rst distinguished,
based on marked differences in time- and voltage-depen-
dent properties (216, 445, 446). Both I
Kr
and I
Ks
are also
coexpressed in guinea pig AVN cells (579). Although I
Kr
activates rapidly, inactivates very rapidly, and displays
marked inward rectication, no inward rectication is
evident for the slowly activating I
Ks
(445, 446). These
channels can also be distinguished at the microscopic
level, as well as by their unique pharmacological proles
(37, 524). Similar to guinea pig, I
Kr
and I
Ks
are also report-
edly coexpressed in human atrial and ventricular myo-
cytes (290, 514, 532, 533), as well as in canine (296, 297,
513, 522, 578) and rabbit (440, 518) ventricles and in
canine Purkinje bers (513) and, in each case, are prom-
inent repolarizing currents. In adult rodent ventricles, in
contrast, I
Kr
and I
Ks
densities are very low (104) or the
currents are undetectable (562).
The unique time- and voltage-dependent properties
of I
Kr
and I
Ks
suggest that these currents play prominent
roles in action potential repolarization, particularly in
ventricular myocytes and Purkinje bers. Nevertheless, in
some cardiac cells, only I
Kr
or I
Ks
appears to be ex-
pressed. In isolated human (225), feline (171), and rat
(404) ventricular myocytes and in rat atrial (404), mouse
SAN (108), rabbit AVN and SAN (218, 233, 467) cells, for
example, only I
Kr
is detected. It has also been reported,
however, that both I
Kr
and I
Ks
are coexpressed in rabbit
SAN cells (284). In this study, the measured densities of
I
Kr
and I
Ks
(in rabbit SAN cells) were quite variable,
although, in general, I
Kr
and I
Ks
densities are highest in
the larger cells found at the periphery of the node (284).
It may be that the densities of I
Kr
and/or I
Ks
in some cells
are too low to be resolved reliably or, alternatively, that
the properties of I
Ks
and I
Kr
in each of these cell types are
distinct from guinea pig ventricular and atrial I
Ks
and I
Kr
.
The apparent absence of I
Kr
and I
Ks
in some cells, as well
as the observation that I
Kr
and I
Ks
expression is hetero-
geneous and variable, might also reect the fact that
functional cardiac Kv channel expression is labile and
might well be affected by the isolation methods, which
typically involve the use of enzymes (578). Detailed stud-
ies focused on current characterizations in intact prepa-
rations, as well as on examining the effects of specic
enzymes and cell isolation methods on Kv current densi-
ties and properties, will be needed to explore these vari-
ous possibilities further.
Although I
Kr
and I
Ks
are not prominent repolarizing
Kv currents in rodent atria or ventricles, there are other
components of delayed rectication with time- and volt-
age-dependent properties distinct from I
Ks
and I
Kr
(Table
1) in myocytes from these (and other) species. In rat
ventricular myocytes, for example, there are multiple de-
layed rectier Kv currents that are coexpressed, and these
are referred to as I
K
, I
Klate
, and I
ss
(26, 210, 561). In adult
mouse ventricular myocytes, three distinct delayed recti-
er Kv currents have also been separated and character-
ized (168, 196, 263, 291, 303, 560, 562, 586, 587), and these
are referred to as I
K,slow1
, I
K,slow2
, and I
ss
(Table 1). Mul-
tiple components of delayed rectication have also been
described in rodent atrial myocytes (68, 69, 74, 75, 497). In
both rat and mouse, it has been demonstrated that all the
various delayed rectier Kv current components contrib-
ute, together with I
to,f
channels, to (ventricular and atrial)
action potential repolarization (291, 303, 560, 586, 587).
It is interesting to note that a steady-state, noninacti-
vating K

current, which resembles I


ss
in rodent atria
and ventricles, has also been described in human atrial
myocytes (58).
In rat (74, 75), canine (577), and human (531533)
atrial myocytes, a novel, very rapidly activating, and
largely noninactivating, outward Kv current, now typi-
cally referred to as I
Kultrarapid
or I
Kur
(479), has been
described (Table 1). In most species, I
Kur
is not detected
in ventricular cells, and it seems likely that the expression
and the properties of I
Kur
, together with I
to,f
, contribute to
determining the more rapid repolarization evident in
atrial, compared with ventricular, myocytes (Fig. 2). Im-
portantly, as in most other species, I
Kur
is not expressed
in human ventricular myocytes or in Purkinje bers, sug-
gesting that I
Kur
channels might represent a therapeutic
target for the treatment of atrial arrhythmias without
complicating effects on impulse propagation, ventricular
functioning, or cardiac output (444). The potential of this
pharmacological strategy, however, will have to be deter-
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mined by the atrial specicity/selectivity of the drugs that
are developed. In contrast to rat, canine, and human, Kv
currents have been described in guinea pig (576) and
mouse (168, 291, 587) ventricular myocytes that have
biophysical properties very similar to human (rat or ca-
nine) atrial I
Kur
. Indeed, the properties of the rapidly
activating, I
Kur
-like, current in guinea pig ventricular myo-
cytes, referred to as I
Kp
(576), and the micromolar 4-AP-
sensitive component of mouse ventricular I
K,slow
, referred
to as I
K,slow1
(69, 291, 302, 587), are indistinguishable from
human (canine and rat) atrial I
Kur
. These currents should,
therefore, probably be renamed I
Kur
(Table 1) to reect
the similarities in properties, as well as molecular identi-
ties of the channels underlying I
Kp
and I
K,slow1
and I
Kur
(see sect. VID).
C. Regional Differences in Kv Current Expression
and Properties
Although the properties of I
to,f
in different cardiac
cells are similar (Table 1), there are marked regional
differences in current densities. In humans (160, 367, 527,
542, 543) and in rats (26, 74, 75), for example, I
to,f
densi-
ties are signicantly higher in atrial, compared with ven-
tricular, myocytes. Similarly, in the rabbit, I
to,s
densities
are higher in atrial myocytes and Purkinje bers than in
ventricular cells (75, 514). In the mouse, however, I
to,f
density is signicantly higher in ventricular (79, 562), than
in atrial (69), myocytes. The density of I
to,f
is also quite
variable in sheep Purkinje bers (520) and in different
regions of the ventricles in canine (294, 297, 522), cat
(178), ferret (77), human (367, 545, 546), mouse (79, 196,
562), and rat (107, 545) hearts. In canine (522) and mouse
(79, 196, 562) heart, for example, I
to,f
density is higher in
the right ventricle (RV), compared with the left ventricle
(LV), and I
to,f
densities are lower in the base of the LV
than in the LV apex (79). In addition, in canine and in
human heart, I
to,f
density varies throughout the thickness
of the ventricular walls, being severalfold higher in the
epicardial and midmyocardial, than in the endocardial,
layers (297, 546). In large mammals, the regional and
cellular heterogeneities in I
to,f
densities are directly re-
ected in the differences in action potential waveforms in
Purkinje, ventricular, and atrial cells (75, 107, 366, 520,
551). Within the ventricles, for example, the differences in
I
to,f
densities are revealed by the presence and the appear-
ance and depth of the notch in the initial phase
(phase 1) of action potential repolarization (24, 364, 374;
see Fig. 2).
There are also marked regional differences in the
expression/distribution of I
to,s
in adult rat, mouse, human,
and canine ventricles (77, 79, 196, 366, 367, 551, 562). In
mouse RV and LV, for example, I
to,s
is undetectable,
whereas cells in the interventricular septum express only
I
to,s
or both I
to,f
and I
to,s
(79, 196, 562). Even when ex-
pressed, however, I
to,f
density is signicantly lower in
septum, compared with ventricular (or atrial), cells (79,
562). The densities of the delayed rectier Kv currents,
I
K,slow1
, I
K,slow2
, and I
ss
, in contrast, are similar throughout
adult mouse ventricles (79, 196, 562). The main deter-
minant of action potential heterogeneity in the mouse,
therefore, appears to be the differential expression of
I
to,f
(79, 196).
In larger mammals, including humans, the differen-
tial expression of I
to,f
is also a primary determinant of
action potential heterogeneity (24, 374). In human heart,
however, it is clear that differences in the expression
levels of the various delayed rectier Kv currents, as well
as the persistent component of the Nav current (see sect.
IIA), also play important roles in regulating action poten-
tial heterogeneity (24, 374). In canine heart, for example,
I
Ks
density is higher in cells in the RV, compared with the
LV, whereas I
Kr
densities are similar in both chambers
(24, 522). I
Ks
density is also higher in canine LV epicardial
and endocardial cells than in M cells (24, 296). In guinea
pig heart, I
Kr
and I
Ks
densities are approximately twofold
higher in atrial, than in ventricular, myocytes (37, 216,
442, 524). There are also regional differences in functional
I
Kr
and I
Ks
expression within the ventricles (80, 316). In
cells isolated from the (guinea pig) LV free wall, for
example, I
Kr
density is higher in subepicardial, than in
either midmyocardial or subendocardial, myocytes (316).
At the base of the LV, however, the densities of both I
Kr
and I
Ks
are signicantly lower in endocardial, than in
either epicardial or midmyocardial, cells (80). These dif-
ferences clearly contribute to the marked differences in
action potential waveforms and frequency-dependent
properties in cells through the thickness of the ventricular
wall (24). In addition to having a major impact on action
potential repolarization, it is now very clear that differ-
ences in functional I
Kr
and I
Ks
densities are also expected
to inuence the maintenance of normal cardiac rhythms
and the susceptibility to rhythm disturbances (24).
IV. OTHER MYOCARDIAL POTASSIUM
CURRENTS CONTRIBUTING
TO REPOLARIZATION
In addition to the depolarization-activated Kv cur-
rents, non-voltage-gated inwardly rectifying K

(Kir) cur-
rents, through I
K1
channels, also contribute to myocardial
action potential repolarization, particularly in ventricular
cells (232, 306, 377, 380). There are also other types of Kir
channels that are expressed and are important in the
normal functioning of the heart, although these do not
seem to play important roles in action potential repolar-
ization under normal physiological conditions (306, 377).
One example of a functionally important class of myocar-
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dial Kir channels is the I
KATP
channels, which are inhib-
ited by intracellular ATP, activated by nucleotide diphos-
phates, and thought to provide a link between cellular
metabolism and membrane potential (232, 380). In the
ventricular myocardium, the opening of I
KATP
channels is
thought to be important under conditions of metabolic
stress, as occurs during ischemia or hypoxia, and to result
in shortening action potential durations and minimizing
K

efux (165, 232). The opening of I


KATP
channels has
also been suggested to contribute to the cardioprotection
resulting from ischemic preconditioning (141, 188). Al-
though I
KATP
channels appear to be distributed uni-
formly in the RV and LV and through the thickness of
the ventricular wall, these channels are expressed at
much higher density than other sarcolemmal K

chan-
nels, suggesting that action potentials could be short-
ened markedly when only very small numbers of I
KATP
channels are activated (463).
Another important cardiac Kir channel type is the
I
K(ACh)
channels, which are gated through a G protein-
coupled mechanism mediated by muscarinic acetylcho-
line receptor activation (275, 564). Physiologically, I
K(ACh)
channels are activated by the binding of G protein -
subunits in response to the acetylcholine released on
vagal stimulation (414). Although I
K(ACh)
channels are
expressed in AVN, SAN, atrial, and Purkinje cells, and are
activated by acetylcholine released on vagal stimulation,
these channels are not thought to contribute appreciably
to action potential repolarization under normal physiolog-
ical conditions. Consistent with this hypothesis, targeted
deletion of one of the Kir subunits (Table 6) encoding
I
K(ACh)
channels, Kir3.4, does not measurably affect rest-
ing heart rates (552). Interestingly, however, atrial bril-
lation is not evident in Kir3.4 null mice exposed to the
acetylcholine receptor agonist carbachol, suggesting that
activation of I
K(ACh)
channels is involved in the cholin-
ergic induction of atrial brillation (269).
As the inward rectier terminology implies, Kir
channels carry inward K

currents better than outward


K

currents (306, 377). Nevertheless, it is the outward K

currents through these channels that are important phys-


iologically because myocardial membrane potentials
never reach values more negative than the K

reversal
potential (approximately 90 mV). As a result, there is
never an opportunity for the inward movement of K

currents through Kir (or any other K

selective) channels.
At the macroscopic level, I
K1
channels have been charac-
terized in human (206, 514), guinea pig (133, 514), and
rabbit (183, 381, 468) atrial and ventricular myocytes and
in rabbit SAN cells (381). The properties of the I
K1
chan-
nels in each of these preparations are similar in that all
are K

selective, blocked by extracellular Ba


2
and intra-
cellular Cs

and strongly inwardly rectifying (183, 206,


223, 514). The strong inward rectication evident in car-
diac I
K1
channels is attributed to block by intracellular
Mg
2
(506), Ca
2
(335), and polyamines (164, 307, 308).
Removal/depletion of intracellular polyamines, Mg
2
and/or Ca
2
, eliminates the steep inward rectication of
(cardiac) I
K1
channels and converts to a linear current-
voltage relation (164, 307, 308, 335, 506).
The expression of I
K1
is clearly reected in the neg-
ative slope region (between approximately 50 and 10
mV) of the (total steady-state) myocyte conductance-volt-
age relation, which is prominent in ventricular myocytes,
but is small or undetectable in atrial cells (183). The fact
that the strongly inwardly rectifying I
K1
channels conduct
at negative membrane potentials suggests that these chan-
nels will play a role in establishing the resting membrane
potentials of Purkinje bers, as well as of atrial and
ventricular myocytes. Direct experimental support for
this hypothesis was provided with the demonstration that
ventricular membrane potentials are depolarized in the
presence of Ba
2
(377), which blocks I
K1
channels. In
addition, action potentials are prolonged, and phase 3
repolarization is slowed in the presence of extracellular
Ba
2
(306), suggesting that I
K1
channels also contribute to
repolarization, particularly in the ventricular myocar-
dium. The voltage-dependent properties of I
K1
channels
(306, 377), however, are such that the conductance is low
at potentials positive to approximately 40 mV. Never-
theless, because the driving force on K

is markedly
increased at depolarized potentials, these channels
should contribute outward K

current during the phase 2


plateau and during phase 3 repolarization (Fig. 2). In
contrast to atrial, ventricular, and Purkinje cells, I
K1
den-
sity is low or undetectable in SAN and AVN cells (244, 381,
468). These observations, as well as the fact that pace-
maker currents are expressed and functional in SAN and
AVN cells, likely explain the ndings that resting mem-
brane potentials in these (SAN/AVN) cells are depolarized
(signicantly) and that the rising phases of the action
potentials in these cells are less steep, relative to resting
membrane potentials/action potentials in atrial and ven-
tricular cells (Fig. 1).
Similar to the Kv channels, I
K1
densities and the
detailed biophysical properties of the currents do vary in
different myocardial cell types. In human heart, for exam-
ple, I
K1
density is more than twofold higher in ventricular,
than in atrial, cells (514). In guinea pig, the properties of
the atrial and ventricular I
K1
currents are also distinct in
that ventricular I
K1
inactivates during maintained depo-
larizations, whereas atrial I
K1
does not (133, 223). In
addition, changes in extracellular K

modulate the mag-


nitude of ventricular I
K1
, but have little effect on atrial I
K1
(223). At the microscopic level, (guinea pig) atrial and
ventricular I
K1
channels are also distinct. Mean channel
open times of ventricular I
K1
channels, for example, are
approximately ve times longer than those of atrial I
K1
channels, whereas the single atrial and ventricular I
K1
channel conductances are indistinguishable (223). Taken
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together, these observations suggested the interesting
possibility that distinct molecular entities underlie ven-
tricular and atrial I
K1
channels, and experimental support
for this hypothesis has now been provided (133).
V. MOLECULAR COMPONENTS OF
MYOCARDIAL NAV AND CAV CHANNELS
A. Nav Channel Pore-Forming -Subunits
Voltage-gated Na

(Nav) channel pore-forming ()


subunits (Fig. 3A) belong to the S4 superfamily of volt-
age-gated ion channel genes (93, 172, 174, 573). Nav -sub-
units have four homologous domains (I to IV), each of
which contains six transmembrane-spanning regions (S1-
S6), and these four domains come together to form the
Na

-selective pore. Structure-function studies have re-


vealed many of the important features of voltage-depen-
dent Nav channel gating (91, 93). The cytoplasmic linker
between domains III and IV, for example, has been shown
to play a pivotal role in voltage-dependent Nav channel
inactivation (392), and a critical isoleucine, phenylala-
nine, methionine (IFM) motif within this linker (91, 444)
has been identied as an important molecular component
of the inactivation gate (516, 517, 544). Voltage-dependent
inactivation of Nav channels is attributed to the rapid
block of the inner mouth of the channel pore by the
cytoplasmic linker between domains III and IV that oc-
curs within milliseconds of membrane depolarization
(483). Consistent with the functional electrophysiological
data, solution NMR analysis of this cytoplasmic linker
peptide revealed a rigid helical structure positioned to
block the pore (427).
Although there are a number of homologous Nav
-subunits (Table 2), Nav1.5 (SCN5A) is the prominent
Nav -subunit expressed in the mammalian myocardium,
and this subunit encodes the rapidly activating and inac-
tivating, tetrodotoxin (TTX)-insensitive Nav channels that
underlie rapid (phase 0) depolarization in atrial and ven-
tricular myocytes and in Purkinje bers (Fig. 1). Never-
theless, several studies have demonstrated that mRNAs
encoding other Nav -subunits, notably Nav1.1, Nav1.3
(120, 426, 471), and Nav1.4 (590), which are typically
considered the Nav -subunits encoding brain and skele-
tal muscle Nav channels, respectively, are also expressed
in the myocardium. In contrast to the Nav channels
formed by Nav1.5, however, Nav1.1-, Nav1.3- and Nav1.4-
encoded Nav channels are blocked by nanomolar concen-
trations of TTX (120, 426, 471, 590). In addition, although
cardiac Nav currents are generally considered relatively
TTX insensitive (174, 573), application of nanomolar con-
centrations of TTX has been reported to shorten canine
Purkinje ber action potential durations (113). These nd-
ings suggest a possible role for TTX-sensitive Nav chan-
nels in the generation of the persistent component of
cardiac Nav currents, at least in canine Purkinje bers.
Nevertheless, there have been very few reports docu-
menting the presence of TTX-sensitive inward Nav cur-
rent components in cardiac cells, raising some concern
about the functional signicance of the expression data,
in spite of the fact that the (message) expression levels of
TABLE 2. Diversity of voltage-gated Na

(Nav) channel - and -subunits


Subfamily Protein Gene Human Mouse Cardiac Current
Nav

1 Nav1.1 SCN1A 2q24 2C1.3 I


Na (TTX)
??
Nav1.2 SCN2A 2q23
Nav1.3 SCN3A 2q24 2C1.3 I
Na (TTX)
??
Nav1.4 SCN4A 17q21 11E1 I
Na (TTX)
??
Nav1.5 SCN5A 3p21 9F3 I
Na (TTX-resistant)
*
Nav1.6 SCN8A 2q13 15F2 I
Na (TTX)
??
Nav1.7 SCN9A 2q24
Nav1.8 SCN10A 3p22 9F3
Nav1.9 SCN11A 3p21 9F3
Nav

X Nav2.1 SCN6A 2q21-23


SCN7A
Nav
1
SCN1B 19p11 7A3 ??

2
SCN2B 11q24 I
Na (TTX-resistant)
*

3
SCN3B 11q26 9F3 I
Na (TTX-resistant)
*

4
SCN4B 11q24
Boxes denote cardiac expression. * Major cardiac (TTX-resistant) Nav current in atria, ventricles, Purkinje bers, and nodal cells. TTX-
sensitive, neuronal-like, Nav current.
1216 JEANNE M. NERBONNE AND ROBERT S. KASS
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Nav1.1, Nav1.3, and Nav1.4 in the myocardium appear to
be quite high (120, 426, 471, 590).
It has also been reported that there are several Nav1
-subunit proteins in addition to Nav1.5 in adult (mouse)
myocardium (314). These include Nav1.1, Nav1.3, and
Nav1.6 (314). The immunolocalization data also suggest
that the Nav1.1, Nav1.3, and Nav1.6 -subunits are local-
ized in the t tubules in adult mouse ventricles (314),
whereas Nav1.5 appears to be localized preferentially to
intercalated disks in mouse, as well as in rabbit and rat,
hearts (270, 315, 400). The subcellular localization of
Nav1.5-encoded myocardial Nav channels at the interca-
lated disks has been interpreted as suggesting that these
(Nav) channels play a major role in regulating conduction
(270). Although the functional role(s) of t-tubular Nav
channels in cardiac functioning has not been established,
voltage-clamp studies have clearly demonstrated that
TTX-sensitive Nav currents can be measured in whole cell
recordings from adult mouse ventricular myocytes
treated with -scorpion toxin, which shifts the voltage
dependence of activation of brain Nav channels, but does
not affect cardiac (i.e., SCN5A-encoded) Nav channels
(314). These observations have been interpreted as sug-
gesting a distinct role for the neuronal Nav channels
localized to the t tubules, i.e., linking depolarization of the
sarcolemmal membrane with the t tubules, thereby cou-
pling depolarization with excitation-contraction coupling
(314). This hypothesis has important functional implica-
tions and certainly warrants further direct experimental
testing.
Immunohistochemical studies have also provided ev-
idence suggesting that Nav1.1 and Nav1.3, but not Nav1.5,
are expressed in rat and mouse SAN (314). These ndings
suggest a substantive molecular difference between the
SAN and the remainder of the myocardium in terms of
Nav channel expression. Given the primary role of the
SAN in regulating heart rate, it would seem certain that
modulating the (TTX-sensitive) Na

current in the SAN


should impact heart rate. Nevertheless, exposure to TTX
reportedly has no effect on heart rate in the mouse (314).
In mice in which one copy of the SCN5A gene has been
disrupted, however, conduction defects, as well as ven-
tricular dysfunction, are evident (389), suggesting that
SCN5A encodes most, if not all, of the cardiac Nav cur-
rent. It seems reasonable to suggest, therefore, that addi-
tional studies focused on exploring the functioning of
Nav1.1-, Nav1.3- and Nav1.6-encoded channels in the
heart are warranted.
During the plateau phase of the action potential in
human ventricular myocytes, 99% of the Nav channels
are in an inactivated, nonconducting state with the inac-
tivation gate occluding the inner mouth of the conducting
pore through specic interactions with sites on either the
S6 segment (345) or the S4-S5 loop (346) of domain IV.
Mutations in the linker between domains III and IV in
SCN5A, linked to the LQT3 syndrome (Fig. 3A), disrupt
Nav channel inactivation (52, 346). These gain of func-
tion mutations lead to an increase in the amplitude of the
sustained component of the Na

current (Fig. 4B) and to


action potential prolongation (Fig. 4A). The enhanced
FIG. 4. Simulated human ventricular action potentials reveal the
impact of gain of function (LQT3) and loss of function (Brugada) mu-
tations in SCN5A. Steady-state action potential waveforms (A and C)
and inward Nav1.5 currents (B and D) were simulated (103, 105). Con-
trol action potential and current waveforms simulated for wild-type
SCN5A-encoded Nav1.5 currents are depicted by the solid black lines in
AD. The corresponding voltage and current waveforms resulting from
LQT3 and Brugada mutations in SCN5A (Fig. 3A) are represented by the
dashed purple (LQT3) and red (Brugada) lines in AD. Gain of function
LQT3 mutations in SCN5A, resulting in an increase in the persistent
component of the Nav current (B), lead to marked action potential
prolongation (A). Loss of function Brugada mutations in SCN5A, in
contrast, result in reduced Nav current (D) and changes in the action
potential upstroke velocity (C). The impact of the loss of function
Brugada mutations are more readily illustrated in the insets of C and D,
in which the voltage (C) and current (D) deections are displayed on an
expanded time scale.
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inward current can be measured during sustained depo-
larizations and appears to reect a change in (Nav chan-
nel) gating that results in channel bursting (52). As
illustrated in Figure 4A, the increase in late inward Na

current (due to Nav channel bursting) prolongs mod-


eled cardiac action potentials (103, 105). Interestingly,
action potential prolongation is also evident in genetically
modied mice expressing human LQT3-associated mutant
SCN5A Nav channels (384). In mice heterozygous for an
SCN5A deletion at residues (KPQ) 15051507, a modica-
tion linked to LQT3, premature ventricular beats and
pacing-induced ventricular tachycardia are also evident
(384). Mutations in SCN5A are also linked to another
(rare) inherited rhythm disorder, the Brugada syndrome
(23, 41) and, similar to LQT3 mutations, a number of
Brugada mutations in SCN5A have been identied (Fig.
4A). In contrast to long QT3, however, Brugada syndrome
mutations are loss of function mutations in Nav1.5 and
result in reduced Nav current (Fig. 4D) and lead to slow-
ing of the action potential upstroke (Fig. 4C). Reductions
in Nav current can also inuence action potential ampli-
tudes (Fig. 4C), as well as phase 1 repolarization. In
addition, owing to intrinsic electrical heterogeneity of the
heart (Fig. 1), the impact of Brugada and LQT3 mutations
in SCN5A would be expected to be variable in different
regions/cell types, an effect which may contribute further
to arrhythmogenesis.
Other identied SCN5A mutations, linked to both the
LQT3 and Brugada syndromes, are found in several other
regions of SCN5A, most notably in the COOH terminus
(Fig. 3A), that also lead to altered channel inactivation
(60, 105, 358, 422, 519, 540). These ndings should prob-
ably have been expected, given that structure-function
studies suggest that multiple domains in Nav -subunits
contribute to the regulation of channel gating (8, 48, 91,
105, 114, 174, 248, 254, 299, 357, 358, 521). A role for the
COOH-terminal tail of Nav1.5 in the regulation of channel
inactivation, for example, has been demonstrated (36, 51,
248, 251, 521). In addition, point mutations in the COOH
terminus affect the kinetics and the voltage dependence
of channel inactivation and recovery from inactivation
and promote sustained channel activity (8, 48, 254, 299,
357). Single-channel studies have also demonstrated that
the proximal portion of the COOH terminus has pro-
nounced effects on repetitive channel openings during
prolonged depolarization (114). Modeling studies of the
COOH terminus of Nav1.5, assuming homology with the
NH
2
terminus of calmodulin, predict that the proximal
region (of the COOH terminus) adopts an -helical struc-
ture, a prediction veried in circular dichroism studies on
a puried COOH-terminal protein (114). The distal region
of the COOH-terminal tail, in contrast, is largely unstruc-
tured and does not appear to affect channel gating mea-
surably (114). These observations suggest that interac-
tions occur between the structured (proximal) region of
the COOH terminus of Nav1.5 and other components of
the channel protein complex and that these interactions
stabilize channels in the inactivated state (114). In addi-
tion, biochemical studies provide strong support for a
model in which there is a direct physical interaction be-
tween the III-IV linker of Nav1.5 and the proximal, struc-
tured portion of the COOH terminus (358). Taken to-
gether, these ndings suggest the formation of a molecu-
lar complex between these domains that is pivotal for
channel inactivation and further that mutations in either
the III-IV linker or the COOH-terminal tail disrupt this
interaction and destabilize inactivation. In addition, the
homology with calmodulin suggests that there may be
structural similarities in the control of Nav and Cav chan-
nel gating, a hypothesis that clearly warrants direct ex-
perimental testing.
Analyses of additional mutations in SCN5A linked to
LQT3, Brugada, and conduction system defects have pro-
vided further molecular insights into Nav channel func-
tioning and arrhythmia mechanisms. One of the well-
described LQT3 mutations, I1768V, for example, does not
result in increased channel bursting, but rather, acceler-
ates the rate of recovery of Nav channels from inactiva-
tion at diastolic membrane potentials (106). Computa-
tional analysis predicted that this mutation would have a
substantial effect under nonequilibrium conditions, e.g.,
during action potential repolarization (106). Subsequent
experiments conrmed this prediction, revealing a novel
mechanism by which mutation-altered Nav channel gating
can prolong cardiac action potentials (7). Interestingly, it
has also been demonstrated that a common polymor-
phism (that results in an S/Y switch) at residue 1102 in
SCN5A is associated with elevated arrhythmia risk in
African Americans (481). Expression studies revealed
that this variant results in very subtle changes in Nav1.5
channel activation and inactivation. Modeling studies sug-
gest, however, that these changes are not likely to alter
cellular electrical activity in carriers unless they are
treated with drugs that block (cardiac) Kv channels (481).
Additional polymorphisms in SCN5A have also been iden-
tied that affect, at least in heterologous expression sys-
tems, the trafcking of functional cell surface Nav chan-
nels (318, 570). In principle, the presence of these poly-
morphisms could, like the S1102Y polymorphism (481),
impact arrhythmia susceptibility in the context of other
factors (e.g., disease or drugs) that affect membrane ex-
citability, action potential durations, and rhythmicity
(318). Mutations in SCN5A and changes in Nav1.5-channel
gating have also been linked to sudden infant death syn-
drome (538). In addition, mutations in two of the neuronal
Nav channel -subunits, SCN1A and SCN2A, are associ-
ated with epilepsies (204, 220, 249, 359). It will be inter-
esting to determine if the molecular mechanisms linking
these mutations, as well as the mutations in the skeletal
muscle Nav channel SCN4A, to disorders in membrane
1218 JEANNE M. NERBONNE AND ROBERT S. KASS
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excitability are similar to those evident for SCN5A in the
LQT3 and Brugada syndromes.
B. Nav Channel Accessory Subunits and Other
Interacting Proteins
Although molecular and functional studies of cardiac
Nav channels have focused primarily on the pore-forming
Nav1.5 -subunit, it is now quite clear that functional Nav
channels in cardiac (and other) cells reect the assembly
of multimeric protein complexes comprising accessory
subunits, as well as a variety of other auxiliary, interacting
and regulatory proteins. All available evidence suggests,
for example, that functional Nav channels in cardiac (and
other) cells are multisubunit proteins consisting of a cen-
tral pore-forming Nav -subunit (Fig. 5) and one to two
auxiliary Nav -subunits (229). In brain, the functional
stoichiometry appears to be one Nav - to two Nav -sub-
units (229); the /-subunit composition of cardiac Nav
changes is probably similar. Three different Nav -subunit
genes, SCN1b (230, 320), SCN2b (231, 241), and SCN3b
(354) encoding Nav1, Nav2 and Nav3 proteins, re-
spectively, have been identied, and it appears that all
three Nav -subunits are expressed in heart (Table 2). The
functional role(s) of the Nav -subunits in the generation
of cardiac Nav currents, however, is not well understood
(20). Expression studies suggest that SCN2b plays a role
in controlling the Ca
2
permeability of Nav channels
(450). The targeted deletion of SCN2b (in Nav2 /
mice) markedly affects neuronal Nav channel expression
and properties and has profound neurological conse-
quences (95). No cardiac phenotype, however, has been
described in Nav2 / mice (95), suggesting that Nav1
or Nav3 more likely contributes to the formation of the
SCN5A-encoded cardiac Nav channels. Consistent with
this hypothesis, heterologous coexpression of Nav1,
which markedly affects Nav1.4-encoded skeletal muscle
Nav channels (319), alters the inactivation kinetics and
the densities of Nav1.5-encoded (cardiac) Nav channels
(20, 134, 155). Heterologous coexpression of SCN3b with
SCN5A also reportedly increases the cell surface density
and modies the inactivation kinetics of Nav1.5-encoded
currents (155).
In addition to modifying the cell surface expression
and the kinetic properties of Nav channels, Nav -sub-
units also appear to be multifunctional cell adhesion mol-
ecules of the IgG superfamily (228) that target channels to
the plasma membrane and mediate channel interactions
with a variety of signaling molecules. It has been demon-
strated, for example, that Nav -subunits interact with
cell adhesion molecules (252, 413), components of the
extracellular matrix (413, 481, 559), and mediate the re-
FIG. 5. Molecular assembly of car-
diac Cav (Nav), Kv, and Kir channels.
Top: the four domains (IIV) of individual
Cav (and Nav) -subunits contribute to
the formation of individual Cav (Nav)
channels, whereas four Kv (or Kir) -sub-
units combine to form tetrameric Kv (or
Kir) channels. Bottom: schematic illus-
trating functional cardiac Nav, Cav, and
Kv channels, composed of the pore-form-
ing -subunits and a variety of channel
accessory subunits.
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cruitment of the actin-binding protein ankyrin to the
plasma membrane at points of cell-cell contact (321). The
ankyrins are (cytosolic) cytoskeletal proteins that have
been suggested to function in regulating the trafcking of
a variety of plasma membrane proteins (53, 54, 350). It has
been demonstrated directly that the intracellular COOH
terminus of Nav1 mediates the interaction with ankyrin
and that Nav1 and ankyrin B associate in transfected
cells and in rat brain membranes (135, 322). Interestingly,
the intracellular COOH-terminal domain of Nav1 has
also been shown to be required for interaction with Nav1,
specically Nav1.2, -subunits (347). Taken together,
these observations suggest an important functional role
for the cytosolic COOH-terminal domain of Nav1 in the
regulation of Nav channel trafcking and/or Nav channel
localization, perhaps through ankyrin B (Fig. 6) and/or
interactions with other components of the actin cytoskel-
eton (135).
It has long been recognized that the proper function-
ing of myocardial Nav channels requires an intact actin
cytoskeleton (324, 504). Disruption of actin polymeriza-
tion on treatment with cytochalasin D, for example, re-
sults in marked (20%) reductions in peak Nav current
densities in isolated rat and rabbit ventricular myocytes
(504). Single-channel recordings from excised membrane
patches from cytochalasin D-treated cells, however, re-
vealed that, in addition to reduced open probability, chan-
nel bursting is increased (504). The latter effect is at-
tributed to a change in Nav channel gating and is func-
tionally similar to the alterations in channel activity seen
with some long QT3 mutations (52, 60, 105, 346, 358, 419,
519, 540). These results suggest the interesting possibility
that multiple pathways (mechanisms) may be important
in mediating Nav channel gating in the normal and in the
diseased myocardium.
An important functional role for the actin cytoskele-
ton in the regulation of Nav channel gating was directly
revealed with the demonstration that mice heterozygous
for a targeted deletion of ankyrin B, ankyrin B /, have
abnormal cardiac electrical activity attributed to altered
Nav channel gating and cell surface expression levels
(40). Single-channel studies on ankyrin B / ventricular
cells revealed increased channel bursting, consistent with
a LQT channel phenotype (94). These observations were
interpreted as suggesting that mutations in ankyrin, which
would lead to Nav channel dysfunction, might well be
important in familial LQT syndromes or other inherited
cardiac rhythm disturbances (50). Consistent with this
hypothesis, molecular genetic studies subsequently re-
vealed a loss-of-function mutation in ankyrin B (E1425G)
that is causally linked to variant 4 of familial long QT
syndrome, LQT4 (351). In addition to providing funda-
mentally important new insights into the molecular defect
underlying LQT4 (351), these ndings demonstrate a
novel, physiologically important, mechanism for regulat-
ing Nav functioning involving interactions between chan-
nel proteins and the cytoskeleton that are coordinated by
the adaptor protein ankyrin B. It is possible that the
ankyrin B mutations interfere directly with Nav1-Nav1
interactions, or perhaps indirectly, through other regula-
tory and/or signaling molecules. In this context, it is also
interesting to note that the cell surface expression of the
Na

-K

-ATPase, the Na

/Ca
2
exchanger, and inositol
1,4,5-trisphosphate (IP
3
) receptors, each of which also
interacts with ankyrin B, are all affected in ankyrin B /
ventricular myocytes (351). It seems certain that the
FIG. 6. Schematic illustrating the
complexity of protein-protein interac-
tions that likely are involved in regulat-
ing/modulating the expression, distribu-
tion, and functioning of myocardial ion
channels. The - and -subunits of Nav
channels interact with the actin cytoskel-
eton through syntrophin-dystrophin and
ankyrin B and with the extracellular ma-
trix through the sarcoglycan complex.
Interactions between Kv channel
(and/or ) subunits and the actin cy-
toskeleton are mediated by the actin
binding proteins lamin and -actinin
and through PDZ domain-containing
scaffolding proteins.
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ankyrin B-mediated interactions between each of these
molecules and the actin cytoskeleton are important for
the normal functioning of each of these ion transport
proteins, as well as the intracellular pathways coupled to
these proteins. The physiological import of these interac-
tions and the impact of the disruption of each of these
interactions on the generation of normal cardiac rhythms
is potentially staggering.
Extrapolating this concept further, it is interesting to
note that there is now a growing body of evidence in the
cardiovascular (and other) system that functional Nav
(and other) channels interact directly and/or indirectly
with the actin cytoskeleton and with a variety of regula-
tory and signaling molecules (Fig. 6) which might well
play a role in the regulation of channel trafcking and
channel expression and/or in the modulation of channel
properties and functioning (418). It has, for example, been
reported that syntrophins, proteins thought to provide a
link between the actin cytoskeleton and other membrane-
associated proteins, interact directly with Nav -subunits,
including Nav1.5 and the skeletal muscle Nav channel
-subunit Nav1.4 (182, 212). Because the syntrophins also
interact directly with dystrophin and dystrobrevin (117)
and, therefore, with the entire dystrophin-associated com-
plex (151) in cardiac and skeletal muscle, it seems rea-
sonable to suggest further that alterations in the proper-
ties of any of the protein components of this macromo-
lecular complex (Fig. 5) could alter myocardial Nav
channel functioning or expression. It is of further interest
to note that cardiomyopathy is often evident in patients
with congenital (skeletal) muscular dystrophies, attrib-
uted to mutations in the dystrophin gene (343), as well as
to mutations in other genes that are part of the dystro-
phin-sarcoglycan protein complex (144). With the as-
sumption that the model of functional cardiac Nav chan-
nels proposed in Figure 6 is at least qualitatively correct,
it would seem reasonable to speculate that the mutations
in any of the genes encoding any of the components of the
depicted macromolecular complex could lead to altered
Nav channel functioning and cardiac arrhythmias alone or
in the background of other myocardial disease, particu-
larly structural heart disease. If complexes such as those
illustrated in Figure 6 are indeed shown to be the physi-
ologically important units of cardiac Nav functioning, ex-
ploring the molecular mechanisms involved in mediating
the many protein-protein interactions important in con-
trolling the assembly, trafcking and functioning of these
macromolecular channel protein complexes might well
provide insights into the link between structural heart
disease and the electrophysiological abnormalities that
are linked to the generation of life-threatening cardiac
arrhythmias in a wide variety of myocardial disease
states.
Other potentially important signaling molecules in
cardiac physiology and pathophysiology are also linked to
syntrophin and, therefore, to Nav1.5 channels (Fig. 6).
Nitric oxide synthase (NOS), for example, which is also
part of the dystrophin-proteoglycan complex (189), and
thought to play a role in myocardial ischemia (466), ap-
pears to bind directly to syntrophin, as well as to caveo-
lin-3 (115, 207, 265), the muscle-specic caveolin in plas-
malemmal caveoli. Nearly all of the endothelial NOS ac-
tivity can be coimmunoprecipitated from cardiac muscle
using an anti-caveolin-3 antibody (161, 162). The caveo-
lins are also important regulators of NOS activity (161,
415), and it is of interest to note that overexpression of
caveolin-3 results in cardiomyopathy and the downregu-
lation of NOS and other components of the dystrophin-
dystroglycan complex (27). Given that it has also been
demonstrated that caveolin-3 binds directly to the cyto-
plasmic tail of -dystroglycan (477), these observations
suggest additional links between cardiac Nav channels,
the actin cytoskeleton, and the extracellular matrix (Fig.
6). Caveolin-3 is thought to play a direct role in regulating
the interaction of caveoli with the sarcolemmal mem-
brane and, therefore, to function to facilitate the transfer
of (Nav or other) channels to the cell surface membrane
from the intracellular compartment (161). It seems rea-
sonable to suggest here that alterations in the interactions
between any of the individual components of the pro-
posed functional Nav channel complex (Fig. 6), through
acquired or inherited disease, could have profound effects
on the properties and/or the functional cell surface ex-
pression of Nav channels, effects that in turn will impact
the generation of normal cardiac rhythms and the likeli-
hood that rhythm disturbances will occur.
In cardiac myocytes, functional cell surface Nav
channel expression is also regulated directly by -adren-
ergic receptor occupancy and the activation of the stim-
ulatory G protein (G
s
) pathway (310, 334). In addition, it
has now been demonstrated that G
s
functions through
binding to caveolin-3, increasing the presentation of
caveoli to the sarcolemmal membrane which could lead
to increased cell surface expression of Nav channels
(569). It has also been reported that caveolin-3 expression
is increased and that nitric oxide signaling is augmented
in a (canine) pacing-induced model of heart failure (203).
The relationship between these biochemical changes and
the observed structural and electrical changes evident in
this model and the relevance of this model and these
changes to human heart failure remain to be established.
As for heterologously expressed Nav channels, there may
well also be additional regulatory molecules, including
growth factors (295, 555) and membrane lipid-anchoring
proteins (460), that play a role in regulating the properties
and the functional cell surface density of cardiac Nav
channels. Clearly, this possibility warrants direct experi-
mental testing.
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C. Cav Channel Pore-Forming -Subunits
Similar to Nav channels, voltage-gated Ca
2
(Cav)
channel pore-forming -subunits belong to the S4
superfamily of voltage-gated ion channel genes (92,
395), and functional voltage-gated Ca
2
(Cav) channels
reect the multimeric assembly of one Cav -subunit
(
1
) and auxiliary Cav and Cav
2
, as well, at least in
some cases, as Cav, subunits (Fig. 5). Also similar to
Nav channels, Cav -subunits comprise four homolo-
gous domains (domains IIV), each of which is com-
posed of six transmembrane segments (S1S6), with an
S4 voltage-sensing domain and a Ca
2
-selective pore
region between S5 and S6 (92, 395). Four distinct sub-
families of Cav channel pore-forming
1
-subunits, Cav1,
Cav2, Cav3, and Cav4 (47), each with many subfamily
members (Table 3), and alternately spliced transcripts
(476) have been identied. The Cav
1
-subunits are
differentially expressed, and studies in heterologous
expression systems have revealed that the various Cav

1
-subunit genes encode Cav channels with distinct
time- and voltage-dependent properties and pharmaco-
logical sensitivities. Heterologous expression of any
one of the four members of the Cav1 subfamily, Cav1.1,
Cav1.2, Cav1.3, or Cav1.4 (Table 3), for example, re-
veals L-type HVA Cav channel currents (92, 395),
whereas heterologous expression of Cav3 -subunits
produces T-type LVA Cav channel currents (92, 395).
Numerous studies, exploiting both heterologous ex-
pression systems in vitro and transgenic strategies in vivo
(363), have provided important (and new) molecular in-
sights into Cav channel composition and functioning in
cardiac (and other) cells. In the past decade, a number of
mutations in Cav channel - and -subunit genes have
also been identied in both humans and mice that result
in disorders of excitability, such as epilepsy, ataxia, peri-
odic paralysis, and migraine (153, 245, 388, 402, 410). Until
very recently, there have been no established links be-
tween inherited disorders of myocardial membrane excit-
ability and mutations in the subunits encoding cardiac
Cav channels. It has now been demonstrated, however,
that a de novo point mutation in the CACNA1C gene,
which encodes the Cav1.2 channel -subunit, underlies
Timothy syndrome, a multisystem disorder with sporatic
inheritance (479). Individuals with Timothy syndrome
have profound cardiac arrhythmias, as well as dysfunc-
TABLE 3. Diversity of voltage-gated Ca
2
(Cav) channel - and -subunits
Subfamily Protein Gene
Locus
Cardiac
Current Subfamily Protein Gene
Locus
Cardiac
Current Human Mouse Human Mouse
Cav

1 Cav1.1 (
1
1.1) (
1S
) CACNA 1S 1q31-32 1E4 Cav
1
CACNB1 17q11.2 11D

2
CACNB2 10p12 2A1 I
Ca(L)
Cav1.2 (
1
1.2) (
1C
) CACNA1C 12p13.3 6E3 I
Ca(L)

3
CACNB3 12q12 15F1

4
CACNB4 2q23 2C1.1
Cav1.3 (
1
1.3) (
1D
) CACNA1D 3p14.3 14A3 Cav
2

2
1 CACNA2D1 7q11.2 5A1 I
Ca(L)
Cav1.4 (
1
1.4) (
1F
) CACNA1F Xp11.23 XA1.1
2
2 CACNA2D2 3p14 9F1 ??

2
3 CACNA2D3 3p13 14A3

2
4 CACNA2D4 12p13
Cav

2
Cav2.1 (
1
2.1) (
1A
) CACNA1A 19p13 8C3
Cav


1
CACNG1 17q26 11E1

2
CACNG2 22q13 15D3
Cav2.2 (
1
2.2) (
1B
) CACNA1B 9q34 2A3
3
CACNG3 16p12 7F2

4
CACNG4 17q26 11E1

5
CACNG5 17q26 11E1
Cav2.3 (
1
2.3) (
1E
) CACNA1E 1q25-31 1G1 ??
6
CACNG6 19q13.4 7A1

7
CACNG7 19q13.4 7A1

8
CACNG8 19q13.4 7A1
Cav

3
Cav3.1 (
1
3.1) (
1G
) CACNA1G 17q21 11D I
Ca(T)
?
Cav3.2 (
1
3.2) (
1H
) CACNA1H 16p13.3 17A3.3 I
Ca(T)
?
Cav3.3 (
1
3.3) (
1I
) CACNA1I 22q13
Boxes denote cardiac expression.
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tion in multiple organ systems, including the immune and
nervous systems (479).
All available molecular and biochemical evidence
suggests that Cav1.2, which encodes the
1C
-subunit, is
the prominent Cav -subunit expressed in the mammalian
myocardium. The CACNA1C gene is large, comprising 44
invariant and 6 alternative exons (474), and the Cav1.2
message is widely expressed. From the many possible
splice sites, a number of different variants of the
1C
protein, including Cav1.2a, Cav1.2b, and Cav1.2c, are gen-
erated (6, 348, 474, 475). Interestingly, although nearly
identical (95%) in amino acid sequence, the various
1C
splice variants appear to be expressed in different cells/
tissues, and the cardiac specic isoform is Cav1.2a, which
encodes cardiac L-type HVA Cav channels (6, 92). Thus
similar to cardiac Nav channels and consistent with the
similarities in the properties of the L-type cardiac Cav
channel currents, it appears that a single pore-forming

1
-subunit (Cav1.2a) is responsible for the generation of
HVA channels throughout the myocardium (92, 395). In-
terestingly, however, it has been reported that sinus node
dysfunction is seen in mice with a targeted disruption
(363) of the Cav1.3 -subunit gene, CACNAID (585), sug-
gesting that SA nodal HVA Cav channels are encoded by
Cav1.3, rather than by Cav1.2 (585). These observations
raise the interesting possibility that, with the right tools,
one would be able to manipulate selectively the function-
ing of atrial/ventricular (Cav1.2) and/or nodal (Cav1.3)-
encoded myocardial Cav channels.
The recently identied linkage between Timothy syn-
drome and a point mutation in the CACNA1C gene encod-
ing Cav1.2a (479) demonstrates, as likely would have been
expected, that defective Cav channel (like defective Na
and Kv channel) functioning can lead to cardiac arrhyth-
mias. The Timothy syndrome mutation is a missense mu-
tation that results in a single amino acid change, glycine
(G) to arginine (R), at residue 406 (479), which is in the
cytoplasmic loop between domains I and II, immediately
C terminal to S6 transmembrane segment in domain I
(Fig. 3). Heterologous expression studies reveal that the
G406R mutation in Cav1.2a markedly reduces voltage-
dependent channel inactivation, resulting in increased
persistent inward Ca
2
current (479). The biophysical
consequence of the Timothy syndrome mutation in
Cav1.2a, therefore, is highly reminiscent of several Nav1.5
channel mutations associated with long QT and Brugada
syndromes that result in increased persistent inward Na

currents (and in action potential prolongation). Interest-


ingly, however, in contrast to the LQT and Brugada syn-
dromes, Timothy syndrome is a multisystem disorder
(479). This latter observation is consistent with the hy-
pothesis that Cav1.2a, unlike Nav1.5, is expressed widely
and that mutations in Cav1.2a result in phenotypic con-
sequences in many different organ systems.
D. Cav Channel Accessory Subunits and Other
Interacting Proteins
There are a number of Cav accessory subunits that
coassemble with Cav
1
(Fig. 5) and play a role in the
generation of functional Cav channels in cardiac, as well
as in other, cells. Three distinct types of Cav channel
accessory subunits, Cav, Cav
2
, and Cav (Table 3), for
example, have been identied (28, 110). Of these subunits,
only the accessory Cav and Cav
2
subunits appear to
be expressed in the myocardium (Table 3) and to contrib-
ute to the formation of functional cardiac Cav channels
(110). The accessory Cav -subunits are cytosolic pro-
teins that assemble with Cav1 -subunits and regulate the
expression of functional cell surface HVA Cav channels,
including cardiac L-type Cav channels. Four different
Cav subunit-encoding genes, CACNB1, CACNB2,
CACNB3, and CACNB4, which encode the Cav
1
(408,
434), Cav
2
(222, 396), Cav
3
(89, 222, 396), and Cav
4
(89, 505) proteins, respectively, have been identied (Ta-
ble 3). It appears, however, that Cav
2
is most promi-
nently expressed in the heart (222, 396). In each Cav
subunit, there are three variable regions anking two
highly conserved domains (89, 222, 396, 408, 434, 505).
The variable regions are in the COOH termini, the NH
2
termini, and a small (100 amino acids) region in the
center of the linear protein sequence between the two
conserved domains (89, 222, 396, 408, 434, 505). The con-
served domains of the Cav -subunits mediate interac-
tions with the pore-forming Cav
1
-subunits, whereas the
variable domains inuence the functional effects of Cav
-subunit coexpression on the properties of the resulting
Cav channels (407). In heterologous expression systems,
coexpression of Cav -subunits with Cav
1
-subunits
markedly increases Cav channel current amplitudes and
densities (59, 187, 541, 565), effects which could reect
increased cell surface channel expression, increased
channel open probability, and/or the stabilization of the
Cav
1
-Cav channel complexes in the cell membrane (98,
99, 565). In addition to increasing current amplitudes,
coexpression of Cav -subunits also modies the kinetics
and the voltage dependences of Cav current activation
and inactivation (70, 242, 279, 353).
A highly conserved sequence motif in Cav
1
-sub-
units, called the alpha subunit interaction domain, ap-
pears to mediate -subunit interaction(s) with accessory
Cav -subunits (407). The Cav
1
interaction domain (Qqx-
ExxLxGYxxWIxxxE) is located in the cytoplasmic loop
between domains I and II, exactly 24 amino acids from the
S6 transmembrane region of domain I (63, 64, 132, 211).
Interestingly, the Timothy syndrome mutation, G406R, is
close to this subunit interaction domain (479), suggesting
that the (G406R) mutations might disrupt Cav
1
-Cav
subunit-subunit interactions. Regions outside of this in-
teraction domain, including low-afnity binding sites in
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the COOH termini of the Cav
1
-subunits, however, have
also been suggested to participate in Cav-Cav
1
subunit-
subunit interactions (412, 493, 523). Indeed, it now ap-
pears that these COOH-terminal regions in Cav
1
-sub-
units also interact specically with a second, highly con-
served domain in the Cav -subunits to produce the
observed modulatory effects of accessory Cav -subunit
coexpression (131, 181).
In addition to the Cav -subunits, another type of
accessory subunit, referred to as Cav
2
, has also been
shown to be part of functional Cav channel complexes
(146). Unlike Cav, the Cav
2
subunits are transmem-
brane accessory subunits (Fig. 5), the rst of which,
Cav
2
-1, was cloned from skeletal muscle (146). At least
four different Cav
2
-1 subunit-encoding genes,
CACNA2D1, CACNA2D2, CACNA2D3, and CACNA2D4,
have been identied (Table 3), and all produce heavily
glycosylated proteins that are cleaved posttranslationally
to yield
2
and proteins that then become linked via
disulde bridges (Fig. 5). In each of the Cav
2
-1 com-
plexes, the Cav
2
domain is located extracellularly,
whereas the Cav domain, which has a large hydrophobic
region, inserts into the membrane (Fig. 5) and serves as
an anchor to secure the entire (Cav
2
) complex (197,
198, 554). In contrast to the accessory Cav subunits, the
functional roles of accessory Cav
2
subunits are variable
and appear to depend, at least in part, on the identities of
the coexpressed Cav
1
and Cav subunits, as well as on
the expression environment. In general, however, coex-
pression of Cav
2
-1 shifts the voltage dependence of
activation of Cav/Cav-encoded channels, accelerates
current activation and inactivation, and increases current
amplitudes, compared with the channels/currents pro-
duced on expression of the Cav
1
and Cav subunits
alone (38, 158, 197, 198, 260, 472). The increase in func-
tional cell surface Cav current densities on coexpression
of Cav
2
-1 (and Cav) subunits appears to reect im-
proved targeting of Cav
1
-subunits to the plasma mem-
brane (469). This effect (improved targeting) is produced
through interactions with Cav
2
, whereas the changes in
channel kinetics are attributed to the presence of the
Cav protein (469).
A distinct type of Cav channel accessory subunit was
revealed with the identication of the Cav subunit,
Cav1, that is expressed in mammalian skeletal muscle,
and that contributes to the formation of functioning of
skeletal muscle Cav channels (462). A number (seven) of
additional Cav-encoding genes, CACNG1-CACNG8 (Ta-
ble 3), have now been identied in skeletal muscle and in
brain (250). All Cav subunits have four transmembrane
spanning domains with the COOH and NH
2
termini pre-
dicted to be intracellular (462). Coexpression of -sub-
units with various combinations of Cav and Cav sub-
units has been shown to affect both the time- and the
voltage-dependent properties of the resulting Cav cur-
rents (250). In addition, the Cav -subunits that are ex-
pressed in the nervous system, Cav
2
, Cav
3
, Cav
4
, and
Cav
8
, all have COOH-terminal PDZ-binding domain mo-
tifs (250). These observations suggest the interesting pos-
sibility that the Cav -subunits play a role in controlling
the localization and/or trafcking of functional Cav chan-
nels (250). Interestingly, it has also been reported that
Cav
2
interacts with the AMPA subtype of neuronal glu-
tamate receptors, suggesting that the Cav -subunits may,
like other channel accessory subunits, be multifunctional
proteins (96). Although Cav -subunits appear to be
widely expressed in the nervous system (250), it is not
clear at present whether one or more of the CACNG genes
is expressed in the heart and/or if these subunits play a
functional role(s) in the generation of cardiac L-type Cav
channels. Clearly, further studies focused on exploring
this topic and determining directly the role(s) of the var-
ious Cav channel accessory subunits in the generation of
cardiac Cav channels are warranted.
As noted in section IIB, it is now very well docu-
mented that HVA myocardial Cav channels undergo rapid
Ca
2
- and voltage-dependent inactivation (44, 166, 281,
326). Fundamentally important insights into the likely
molecular mechanism underlying the Ca
2
-dependent
component of inactivation were revealed with the dem-
onstration that the EF-hand domain containing protein,
calmodulin, that binds Ca
2
and modulates a variety of
Ca
2
-dependent processes, is associated with the COOH-
terminal cytoplasmic domain of L-type HVA channels
(398). Several subsequent studies have provided many of
the molecular details of the calmodulin:Cav channel
1
-
subunit association and interaction domains (17, 149, 293,
355, 428). In addition, the generality of the calmodulin-
mediated mechanism of Ca
2
-dependent inactivation of
Cav channels was documented with the demonstration
that P/Q-type neuronal HVA channels are also regulated
by calmodulin binding (128).
In addition to the regulation of channel gating by
Ca
2
and calmodulin, the properties and the functional
expression of myocardial Cav channels are also regulated
by a variety of extracellular signals and intracellular sig-
naling pathways. Prominent among these are the rather
well studied -adrenergic G protein-coupled receptor-me-
diated augmentation of cardiac L-type Cav channel cur-
rents, increased Ca
2
entry, and positive inotropy (255,
509). Considerable experimental evidence suggests that
the pore-forming Cav1.2 -subunit and Cav -subunits are
targets of posttranslational modications by a variety of
protein kinases that impact the functional cell surface
expression and the properties of cardiac L-type Cav chan-
nels (255, 509). It has also been reported that cardiac HVA
Cav channels are actually associated with -adrenergic
receptors in macromolecular complexes that likely also
include heterotrimeric G proteins, adenylate cyclase, pro-
tein kinases, phosphatases and protein kinase A binding
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proteins, or AKAPs (18, 122), which appear to subserve a
scaffolding function (18). Unlike Nav channels, however,
no direct links between cardiac Cav channel subunits and
actin or actin-binding proteins have been demonstrated to
date. Nevertheless, a number of Ca
2
-binding proteins,
including calmodulin, have been shown to be linked both
directly and indirectly to the actin cytoskeleton (452). In
addition, it has been reported that the time- and voltage-
dependent properties of L-type HVA channels are altered
in skeletal muscle from dystrophin-decient animals, an
effect interpreted as resulting from remodeling of the
subcellular actin cytoskeleton (111). Similarly, neuronal
HVA Cav channel inactivation is differentially affected by
agents that stabilize and destabilize the actin cytoskeleton
(239). In retinal ganglion neurons, for example, stabiliza-
tion or disruption of the actin cytoskeleton affects func-
tional cell surface Cav channel expression (454). It seems
reasonable to suggest, therefore, that Cav channels inter-
act directly or indirectly with the actin cytoskeleton (556)
and that, similar to cardiac Nav channels, the functioning
of myocardial Cav channels likely also depends impor-
tantly on interactions with the actin cytoskeleton. In sup-
port of this hypothesis, targeted deletion of endothelial
NOS eliminates the muscarinic modulation of myocardial
L-type Cav channels (202). Further investigations focused
on delineating the molecular mechanism controlling func-
tional Cav channel expression will be needed to dene the
role of the cytoskeleton in regulating myocardial Cav (and
Nav) channel expression and functioning.
VI. MOLECULAR COMPONENTS OF
MYOCARDIAL KV CHANNELS
A. Kv Channel Pore-Forming -Subunits
Similar to Nav and Cav -subunits, voltage-gated K

channel (Kv) pore-forming () subunits (Fig. 3) belong to


the S4 superfamily of voltage-gated channels (405). In
contrast to Nav and Cav channel -subunits, however, Kv
-subunits are six transmembrane-spanning domain pro-
teins (Fig. 3, B and C), and functional Kv channels com-
prise four -subunits (Fig. 5). A very large number of Kv
-subunit genes have been identied, and a systematic
terminology for naming these subunits (Table 4) has been
developed (199). Heterologous expression of Kv -sub-
units in the Kv1-Kv4 subfamilies reveals functional Kv
channels with distinct time- and voltage-dependent prop-
erties (116), whereas Kv -subunits of the Kv59 subfam-
ilies (Table 4) are electrically silent (88, 142, 221, 441).
Coexpression of Kv5-Kv9 subunits with Kv2 -subunits,
however, attenuates the amplitudes of the Kv2-encoded
K

currents (441). Nevertheless, the functional roles of


the silent Kv -subunits in the generation of myocardial
Kv channels remains to be determined.
Further functional Kv channel diversity in cardiac
and other cells could, in principle, arise through alterna-
tive splicing of transcripts (29), as well as through the
formation of heteromultimeric channels (116) between
two or more Kv -subunit proteins in the same Kv sub-
family. Kv channel assembly, as well as the properties of
the resulting channels, are largely determined by the in-
tracellular NH
2
- and COOH-terminal -subunit domains
(100). Molecular and biochemical studies have revealed
that, of the many Kv1-Kv9 -subunits identied, only a
small subset is expressed in the heart (Table 4). Although
many studies have characterized the detailed time- and
voltage-dependent properties of the various Kv -subunit-
encoded Kv channels in heterologous expression systems,
these studies have provided little insight into the molec-
ular correlates of functional cardiac Kv channels. The
difculties encountered in these studies probably reect
the fact that Kv channel properties depend on the expres-
sion environment (397), likely owing to cell-type specic
differences in posttranslational processing of the Kv
channel -subunit proteins and/or the expression of Kv
channel accessory subunits or other Kv channel interact-
ing, regulatory proteins (397).
Additional subfamilies of Kv -subunit genes in the
KCNQ and KCNH subfamilies (Table 4) have been iden-
tied, and one member of each of these subfamilies,
KCNQ1 and KCNH2, has been shown to be the loci of
mutations leading to congenital long QT syndromes, LQT1
(Fig. 3C) and LQT2 (Fig. 3B), respectively (40, 119, 447,
448, 499, 528). Heterologous expression of human
KCNH2, which encodes the ether-a-go-go-related protein
ERG1, reveals Kv currents (448, 499) that are similar to
cardiac I
Kr
(Table 4). Similar to SCN5A mutations linked
to the LQT3 and Brugada syndromes (Fig. 3A), LQT2
mutations in KCNH2 are found throughout the ERG1
protein sequence (Fig. 3B). These (LQT2) mutations are
all loss of function and result in reduced functional I
Kr
channel expression owing to dominant negative effects or
to alterations in channel processing or trafcking (23, 126,
220, 246, 253, 273, 584). Interestingly, a novel gain of
function mutation in KCNH2, which results in increased
I
Kr
channel densities, has recently been identied and
linked to one form of short QT syndrome (78).
There are six additional members of the KCNH sub-
family, KCNH3-KCNH8 (Table 4). Two of these, KCNH3
and KCNH4, appear to be nervous system specic (465),
and it is presently unclear whether any of the others are
present in the myocardium. Alternatively processed forms
of KCNH2, with unique NH
2
and COOH termini, however,
have been cloned from both mouse and human heart
cDNA libraries and postulated to contribute to the gener-
ation of functional cardiac I
Kr
channels (272, 282, 304).
Indeed, coexpression of the NH
2
-terminal splice variant
ERG1b with the full-length ERG1a produces Kv currents
that more closely resemble cardiac I
Kr
than the currents
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produced on expression of ERG1a alone (304). Although
it has been reported that only the full-length ERG1 pro-
tein(s) are detected in adult rat, mouse, and human hearts
(404), raising some doubts about the functional signi-
cance of alternative splicing of KCNH2 transcripts, more
recent studies have identied ERG1b protein in adult rat,
human, and canine heart (240). Presumably, these dispar-
ate results reect the fact that different anti-ERG1 anti-
bodies were used (240, 404). Further studies will be
needed to explore this and other possible explanations.
Using antibodies targeting the specic ERG1 isoforms, it
was also demonstrated that ERG1a and ERG1b coimmu-
noprecipitate from heart, suggesting that functional car-
diac I
Kr
channels reect the heteromeric assembly of
ERG1a and ERG1b subunits (240). The expression, distri-
bution, and functioning of the COOH-terminal variant of
ERG, ERG-USO (272), in contrast, remains to be explored.
As noted previously, mutations in the KCNQ1 gene
have been linked to LQT1 (253, 329). Heterologous ex-
pression of KvLQT1 (KCNQ1) alone yields rapidly acti-
vating and noninactivating outward Kv currents, whereas
coexpression with the Kv channel accessory subunit,
TABLE 4. Diversity of voltage-gated K

(Kv) channel -subunits


Subfamily Protein Gene
Locus
Cardiac
Current Subfamily Protein Gene
Locus
Cardiac
Current Human Mouse Human Mouse
Kv1 Kv8
Kv1.1 KCNA1 12p13 6F2 Kv8.1 KCNV1 8q21.1
Kv1.2 KCNA2 1p11 3F2.2 I
K,slow
(I
K,DTX
)
Kv9
Kv8.2 KCNV2 9p24
Kv1.3 KCNA3 1p21 3F2.2 Kv9.1
Kv9.2
KCNS1
KCNS2
20q12 2H3
Kv1.4 KCNA4 11p14 2E2 I
to,s
Kv9.3 KCNS3 2p25 15B3.1
Kv1.5 KCNA5 12p13 6F2 I
Kur
I
K,slow1
eag
Kv1.6 KCNA6 12p13 6F2 eag KCNH1 1q32 1H6
Kv1.7 KCNA7 19q13 7B3 erg1 KCNH2 7q36 5A3 I
Kr
Kv1.10 KCNA10 1p11 erg2 KCNH3 15
erg3 KCNH4 17q21
Kv2 erg4 KCNH5 14q22
Kv2.1 KCNB1 20q13.1 2H3 I
K,slow2
erg5 KCNH5 17q24
Kv2.2 KCNB2 8q13 ?? erg6 KCNH7 2 2C1.1
erg7 KCNH8 3p24
Kv3 KvLQT
Kv3.1 KCNC1 11p15 7B3 I
Ks
Kv3.2 KCNC2 12q21 KvLQT1 KCNQ1 11p15 7F6
Kv3.3 KCNC3 19q13.4 7B2 KCNQ2 KCNQ2 20p11.1 2H4
Kv3.4 KCNC4 1p11 3F2.2 KCNQ3 KCNQ3 8q24.3 15D1
Kv4 KCNQ4 KCNQ4 1p34.3
Kv4.1 KCND1 Xp11.2 ?? KCNQ5 KCNQ5 6q11
Kv4.2 KCND2 7q32 6A2 I
to,f
Kv4.3 KCND3 1p11 3F2.2 I
to,f
Kv5
Kv5.1 KCNF1 2p25 ??
Kv6
Kv6.1 KCNG1 20q13.1
Kv6.2 KCNG2 18q23
Kv6.3 KCNG3 2p21 17E3
Kv6.4 KCNG4 16q24
Boxes denote cardiac expression.
1226 JEANNE M. NERBONNE AND ROBERT S. KASS
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minK (see sect. VB), produces slowly activating K

cur-
rents that resemble the slow component of cardiac de-
layed rectication, I
Ks
(40, 447). Similar to the SCN5A
mutations linked to the LQT3 and Brugada syndromes
(Fig. 3A) and KCNH2 mutations linked to LQT2 (Fig. 3B),
KCNQ1 mutations linked to LQT1 have been identied
throughout the protein sequence (Fig. 3C). Expression
studies suggest that the various LQT1-associated muta-
tions in KCNQ1 are all loss of function, resulting in re-
ductions in functional I
Ks
cell surface channel expression.
Simulations demonstrate that reductions in I
Ks
density
(Fig. 7B) result in markedly prolonged ventricular action
potential waveforms (Fig. 7A). Given the intrinsic heter-
ogeneities in I
Ks
(and other) channel densities and action
potential waveforms throughout the myocardium (Fig. 1),
the effects of LQT1 mutations in KCNQ1 might also be
heterogeneous, further impacting the arrhythmogenic po-
tential of these mutations. Importantly, a novel gain of
function mutation in KCNQ1 (V307L) was identied and
linked to short QT interval syndrome (46). Heterologous
expression studies revealed that expression of KCNQ1
V307L, alone or with wild-type KCNQ1, in the presence of
KCNE1, produces I
Ks
-like currents with markedly altered
activation kinetics and voltage-dependent properties (46)
relative to the channels produced by wild-type KCNQ1
and KCNE1. A gain of function mutation (S140G) in
KCNQ1 has also been identied in a family with heredi-
tary persistent atrial brillation (97). Computer simula-
tions incorporating the KCNQ1 short QT mutant channels
reveal that I
Ks
densities are increased (Fig. 7D) and that
action potentials are shortened markedly (Fig. 7C). As
noted above for LQT1 mutations, the impact of gain of
function mutations in KCNQ1 on different cell types and
regions of the heart will likely be heterogeneous, owing to
the existing heterogeneity in I
Ks
(and other current) den-
sities and action potential waveforms, an effect which
may acerbate the arrhythmogenic potential of alterations
in I
Ks
densities.
Similar to the multiplicity of -subunits in the Kv and
the KCNH subfamilies, there are a number (four) of ad-
ditional members of the KCNQ subfamily (Table 4), al-
though none of these appears to be expressed in heart.
Two of the KCNQ subfamily members, KCNQ2 and
KCNQ3, however, are expressed in the nervous system
and have been identied as loci of mutations leading to
benign familial neonatal convulsions (65, 329, 453, 526).
Heterologous expression of KCNQ2 or KCNQ3 results in
the generation of slowly activating, noninactivating K

-
selective channels that also deactivate very slowly on
membrane repolarization (329, 453, 526). Interestingly,
the properties of the heteromeric KCNQ2/KCNQ3 chan-
nels closely resemble neuronal muscarinic acetylcholine
receptor regulated ion channel currents, typically referred
to as M currents/channels (329, 526).
B. Kv Channel Accessory Subunits
Similar to the Nav and Cav channels, a number of
different types of Kv channel accessory subunits have
been identied (Table 5) and postulated to contribute to
the generation of functional myocardial Kv channels. The
FIG. 7. Simulations reveal the effects of loss of function (LQT1) and
gain of function (short QT) mutations in KCNQ1. Steady-state action
potential waveforms (A and C) and outward I
Ks
currents (B and D) were
simulated (103, 105). Control action potential and current waveforms
simulated for wild-type KCNQ1-encoded I
Ks
currents are depicted as the
solid black lines in AD. The corresponding simulated voltage and
current waveforms depicting the effects of KCNQ1 mutations are illus-
trated by the dashed purple (LQT1) and red (short QT) lines in AD.
Loss of function LQT1 mutations (Fig. 3C), resulting in a decrease in
the maximum amplitude and a slowing of the time to peak of I
Ks
(B),
lead to marked action potential prolongation (A). In contrast, gain of
function short QT mutations in KCNQ1 increase the maximal amplitude
of I
Ks
(D) and shorten action potential durations.
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rst of these was cloned from human (362), and later from
rat (170), heart and was referred to as minK, i.e., minimal
K

channel subunit. MinK, which is encoded by the


KCNE1 gene on chromosome 21 in human (Table 5), is a
small (130 amino acids) protein with a single transmem-
brane spanning domain (170, 288, 362). Although initial
characterizations of minK in Xenopus oocytes suggested
that this small protein could produce functional Kv chan-
nels when expressed alone (170, 362), subsequent studies
demonstrated the presence of a KCNQ1 homolog in oo-
cytes that combines with the heterologously expressed
minK to generate Kv channels that very closely resemble
cardiac I
Ks
(447). As noted above, these observations have
led to suggestions that minK coassembles with the Kv-
LQT1 protein to produce functional cardiac I
Ks
channels
(40, 447). It has also been reported, however, that minK
coassembles with the ERG1 protein in heterologous ex-
pression systems, observations interpreted as suggesting
a role for minK in the generation of cardiac I
Kr
channels
(341). It has become increasingly clear, however, that
accessory Kv subunits, such as minK, can, at least in
heterologous expression systems, associate with multiple
different Kv subunits. It is not a given, however, that
these interactions occur in intact tissues. As a result, it is
presently unclear whether minK subunits actually associ-
ate with both KvLQT1 and ERG1 (and other Kv?) -sub-
units in the myocardium and contribute to the function of
both cardiac I
Ks
and I
Kr
(and other Kv?) channels. Bio-
chemical studies focused on exploring these questions
are clearly warranted. Although the details of minK func-
tioning remain to be claried, it is important to emphasize
that the physiological signicance of this subunit in the
generation of cardiac membrane currents was clearly
demonstrated with the identication of mutations in
KCNE1 that are associated with one type of inherited long
QT syndrome, LQT5 (62, 478, 481).
Several additional members of the minK-related pep-
tide, MiRP (KCNE), subfamily (Table 5) have also been
identied and characterized in coexpression studies with
Kv -subunits (13, 339). One of these, MiRP1 (KCNE2),
has been suggested to function as an accessory subunit of
ERG1 (KCNH2) to generate cardiac I
Kr
(1, 5). As noted
above, however, it has previously also been suggested
that minK associates with ERG1 to produce I
Kr
channels
(341). Although the resolution of this seeming contro-
versy must await further experimentation, particularly
biochemical studies, it is reasonable to conclude that
MiRP1 (KCNE2) is functionally important in the regula-
tion of myocardial membrane excitability, as evidenced
by the fact that KCNE2 variants are associated with spo-
radic and drug-induced long QT syndromes (5, 227, 457,
478). It is also interesting to note that studies in heterol-
ogous systems have revealed that the MiRP subfamily of
Kv channel accessory subunits can assemble with Kv
-subunits in several different subfamilies (4, 583) to
modify the properties and/or the cell surface expression
of Kv -subunit-encoded channels. Heterologous expres-
sion studies, for example, have shown that MiRP1 also
interacts with Kv4 -subunits (583). In addition, it has
been demonstrated that MiRP2 (KCNE3) forms Kv chan-
nels with Kv3.4 -subunits in skeletal muscle and that
mutations in MiRP2 result in reduced Kv3.4-encoded Kv
current densities, membrane depolarization, and periodic
paralysis (4). Biochemical and coexpression studies have
also suggested that MiRP1 can associate with hyperpolar-
ization-activated cyclic nucleotide-gated (HCN) cation
channels, suggesting a distinct function for the MiRP1
subunit in the regulation of myocardial pacemaker cur-
rents, rather than, or in addition to, the regulation of
myocardial Kv channels (574). Taken together, these ob-
servations suggest the interesting possibility that mem-
bers of the KCNE subfamily might be multifunctional
proteins, coassembling with several different Kv (3)
and/or other (e.g., HCN) ion channel pore-forming -sub-
units and contributing to the formation of multiple types
of cardiac Kv (and other) channels. Experiments focused
on testing this hypothesis and on dening the functional
roles of the various MiRP (KCNE) subunits in the gener-
ation of myocardial Kv (and other) channels will be of
considerable interest.
Another type of Kv channel accessory subunit was
revealed with the biochemical identication (360) and
TABLE 5. Auxiliary Kv channel subunits
Family Subunit Gene Human Mouse Current
Kv
Kv1 KCNAB1 3q25 3D ??
Kv2 KCNAB2 1p36.3 4E2 ??
Kv3 KCNAB3 17p13 11B3
KCNE
Mink KCNE1 21q22 16C4 I
Ks
MiRP1 KCNE2 21q22 16C4 I
Kr
??, I
to,f
??
MiRP2 KCNE3 11q13 7E1 ??
MiRP3 KCNE4 2q36.3
MiRP4 KCNE5 Xq22 XF1
KChAP
KChAP PIAS3 1q12 3F1 I
to,f
??, I
K
??
KChIP
KChIP1 KCNIP1 5q35 11A4
KChIP2 KCNIP2 10q25 19C3 I
to,f
, others??
KChIP3 KCNIP3
KChIP4.2 CSEN 2q11.1 2F1
KChIP4.3 KCNIP4 4p15.3
NCS
NCS-1 FREQ 9q34 2A3 I
to,f
, others??
Boxes denote cardiac expression.
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subsequent cloning (419) of low molecular mass (45
kDa) cytosolic Kv -subunits from brain. Three distinct,
but homologous, Kv -subunits, Kv 1, Kv 2, and Kv 3,
encoded on three different (KCNAB) genes (Table 5), as
well as alternatively spliced transcripts (337), have now
been identied (90, 124, 147, 148, 317, 352). Of these,
Kv1.1, Kv1.2, Kv1.3, and Kv2 have been shown to be
expressed in the heart (12, 90, 124, 147, 148, 278, 317, 352).
The Kv -subunits share a conserved COOH-terminal
core region, related in amino acid sequence to aldo-
ketone reductases, members of the triose phosphate
isomerase enzyme family (101, 338). The NH
2
-terminal
domains of the Kv -subunits are unique, and members of
the Kv1 subfamily, as well as Kv3.1, have long NH
2
-
terminal sequences that are structurally similar to the
Shaker (Kv1) channel inactivation gate that functions in a
ball and chain-like mechanism (217) to accelerate Kv1
channel inactivation (300 301). Although the core region
of the Kv -subunits contains an NAPH/NADPH binding
site (101, 338) and Kv -subunits crystallize with NADPH
bound (191), the role of this binding in regulating the
functional interactions between Kv - and Kv -subunits
and/or in controlling the expression/properties of the re-
sulting Kv channels has not been dened.
Previous studies have demonstrated that the Kv
-subunits interact with the intracellular T1 tetrameriza-
tion domain of the Kv -subunits of the Kv1 subfamily,
combining in a 1:1 stoichiometric ratio (190, 192). Heter-
ologous coexpression studies have revealed that Kv
-subunits affect the functional properties and the cell
surface expression of Kv1 -subunit-encoded channels (9,
10, 90, 147, 148, 317, 352, 464). In some cases, the func-
tional consequences of Kv coexpression have been
shown to be Kv1 -subunit specic (10). Coexpression of
Kv2, for example, increases the expression of Kv1.2-
encoded channels and decreases the expression of Kv1.5-
encoded channels (9, 10). Because Kv1 and Kv sub-
units coassemble in the endoplasmic reticulum (369), the
observed increases in functional cell surface Kv1-encoded
channel expression suggest that the Kv subunits affect
Kv1 channel assembly, processing or stability or, possibly,
function as chaperone proteins.
The facts that the Kv -subunits were originally iden-
tied in association with Kv1 -subunits (360) and that
heterologous expression studies suggest that Kv1 and
Kv2 interact only with the Kv1 -subunits (370, 458)
have been interpreted as suggesting that the Kv -sub-
units function as specic accessory subunits in the gen-
eration of Kv channels encoded by Kv1 -subunits. In
expression systems, however, Kv3 has also been shown
to interact specically with Kv2 subunits (167). Biochem-
ical studies also suggest that Kv -subunits might well be
functionally more diverse (370, 393, 458). Both Kv1.1 and
Kv1.2, for example, coimmunoprecipitate with Kv4
-subunits following coexpression in COS-1 cells (370).
Coexpression of Kv1 subunits has also been reported to
modulate the cell surface expression of Kv4.3-encoded
currents, an effect attributed to Kv1 binding to the
COOH terminus of Kv4.3 (566). In addition, although with-
out any detectable effect on current properties or densi-
ties, coexpression (in Xenopus oocytes) of Kv1.2 with
Kv4.2 -subunits modulates the redox sensitivity of Kv4.2-
encoded channels (393).
Similar to the KCNE subfamily of Kv accessory sub-
units, therefore, the functional roles assigned to the Kv
-subunits in the generation of myocardial Kv channels
have been largely a matter of speculation. Interestingly,
however, recent studies, completed on ventricular myo-
cytes isolated from mice with a targeted disruption in the
Kv1 locus, reveal that the loss of Kv1 affects the func-
tional cell surface densities of Kv4- and Kv2-encoded, but
not Kv1-encoded, Kv channels (12). It has also been re-
ported that Kv -subunits associate with -subunits of the
eag (KCNH) subfamily (553). On the basis of all the
available data, therefore, it seems reasonable to suggest
that the Kv -subunits subserve multiple functions in the
generation of myocardial Kv channels. Further studies
focused on providing the molecular details of Kv -func-
tioning are clearly warranted.
A novel Kv channel accessory protein, KChAP (K

channel accessory protein) (Table 5), was identied in a


yeast two-hybrid screen using the NH
2
terminus of Kv4.2
as the bait (550). Sequence analysis of KChAP revealed a
574-amino acid protein with no transmembrane domains
and no homology to Kv - or Kv -subunits (550). Coex-
pression of KChAP with Kv2.1 (or Kv2.2) in Xenopus
oocytes, however, markedly increases functional Kv2.x-
induced current densities without measurably affecting
the time- and/or voltage-dependent properties of the cur-
rents (550), suggesting that KChAP functions as a chap-
erone protein (277). Yeast two-hybrid assays also re-
vealed that KChAP interacts with the NH
2
termini of
-subunits in the Kv1 subfamily and with the COOH ter-
mini of Kv 1-subunits (278, 550). Interestingly, it was
subsequently demonstrated that KChAP belongs to the
protein inhibitor of the activated STAT family of proteins
that interact with a variety of transcription factors and
play a role in programmed cell death (549). The relation-
ship between the apoptotic and chaperone functions of
KChAP, as well as the functional role of KChAP in the
generation/regulation of Kv channels in the normal and
the diseased myocardium, however, remain to be deter-
mined.
With the use of the intracellular NH
2
terminus (amino
acids 1180) of Kv4.2 as the bait in a yeast two-hybrid
screen, three novel Kv channel interacting proteins,
KChIP1, KChIP2, and KChIP3 (Table 5), were identied
(21). The KChIPs belong to the recoverin family of neu-
ronal Ca
2
-sensing (NCS) proteins, particularly in the
core regions, which contain multiple EF-hand domains
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(81). Unlike other NCS-1 proteins, however, KChIP2 and
KChIP3 lack NH
2
-terminal myristoylation sites, and the NH
2
termini of each of the KChIP proteins are unique (21). Nev-
ertheless, KChIP2 and KChIP3 have several potential palmi-
toylation (on cysteine residues) sites, and metabolic labeling
studies suggest that these sites are palmitoylated in situ
(491). It is now clear that KChIP3 is identical to the previ-
ously identied protein calsenilin, which is a Ca
2
-binding
protein that interacts with presenilin-1 and presenilin-2 and
regulates the proteolytic processing of these two proteins
(82). In addition, however, KChIP3 is also identical to an-
other previously identied protein called DREAM, which is
a Ca
2
-regulated transcriptional repressor (87). The DREAM
protein has been shown to bind to the downstream regula-
tory elements (DRE) of several genes in the absence of Ca
2
and to dissociate from the DRE sequence when Ca
2
is
elevated (87). Thus DREAM is thought to act as an activity-
dependent regulator of gene expression (87). An additional
member of the KChIP family, KChIP4, also referred to as
calsenilin-like-protein or CALP, was subsequently identied
in biochemical studies focused on identifying the binding
partners of the presenilin proteins (356). The interactions
between KChIP3 (calsenilin) and KChIP4 (CALP) and the
presenilin proteins are also Ca
2
dependent (356).
Of the four KChIP genes, only KChIP2 appears to be
expressed in the heart (21, 431). There are, however,
numerous splice variants of KChIP2 that have now been
identied (33, 125, 129, 390, 391, 430, 431). Studies in
heterologous systems have revealed that coexpression of
any one of the (full-length) KChIP proteins with Kv4
-subunits increases the functional cell surface expres-
sion of Kv4.x-encoded Kv channels, slows current inacti-
vation, speeds recovery from inactivation and shifts the
voltage dependence of channel activation (21, 193, 195).
In contrast, KChIP expression reportedly does not affect
the properties or the densities of the K

currents pro-
duced on expression of other Kv -subunits, including
Kv1.4 and Kv2.1 (21). These observations were inter-
preted as suggesting that the modulatory effects of the
KChIP proteins are specic for -subunits of the Kv4
subfamily (21). In addition, although the binding of the
KChIP proteins to Kv4 -subunits is not Ca
2
dependent,
mutations in EF hand domains 2, 3, and 4 of KChIP1
reportedly eliminate the modulatory effects of KChIP1 on
Kv4.2-encoded K

currents in CHO cells (21). It has,


however, also been reported that a splice variant of
KChIP2, KChIP2d, which lacks three of the four EF hand
domains of full-length KChIP2, modies the inactivation
kinetics of heterologously expressed Kv4.3-encoded K

currents, but does not alter the kinetics of channel recov-


ery from steady-state inactivation (390). Taken together,
these ndings suggest that distinct regions of the (full-
length) KChIP proteins underlie the various modulatory
effects of KChIPs on the properties and cell surface ex-
pression of Kv4-encoded channels. Structural analysis has
revealed that Kv4 and KChIP accessory subunits assem-
ble in a 4:4 stoichiometry and provide new insights into
the intracellular interactions between the NH
2
termini of
Kv4 subunits and the EF hand domains of the KChIPs
(258, 588). In addition, it had been shown that myristoyl-
ation of KChIP1 appears necessary for the normal traf-
cking of newly synthesized (KChIP1) protein to the en-
doplasmic reticulum where the association with Kv4
-subunits occurs (385). It may be that palmitoylation of
KChIP2 and KChIP3 plays a similar functional role. Mu-
tagenesis and structural studies have also revealed that
two regions in the NH
2
termini of Kv4 subunits are nec-
essary for KChIPx interaction with (and modulation of)
Kv4-encoded channels and that residues 7190 (in Kv4.x)
form a contact loop that mediates the interaction with
the KChIP protein(s) (451).
Biochemical methods were exploited in efforts that
led to the identication of another Kv channel accessory
subunit, DPPX or DPP6, that also appears to interact
specically with Kv4 -subunits (368). A novel protein of
previously unknown function, DPP6 is structurally related
to CD26, which is a dipeptidyl aminotransferase and a cell
adhesion protein (368). Interestingly, DPP6 actually be-
longs to a family of nonclassical serine proteases, al-
though DPP6 itself has no enzymatic activity (368). In
contrast to the KChIPs, DPP6 is an integral membrane
glycoprotein with a rather large extracellular COOH-ter-
minal domain (368). Coexpression of DPP6 with Kv4
-subunits affects the trafcking and the membrane tar-
geting of Kv4 -subunits and modies the kinetic proper-
ties of expressed cell surface Kv4-encoded channels
(368). Although expressed in brain and thought to func-
tion in the generation of neuronal Kv4-encoded transient
outward Kv currents, DPP6 does not appear to be ex-
pressed in heart and, therefore, cannot contribute to the
formation of functional cardiac Kv channels. Another
member of the dipeptidyl transferase family, DPP10, has
also been shown to associate with Kv4 -subunits in
heterologous expression systems and to modify the bio-
physical properties of Kv4.x-encoded channels (235). The
effects of DPP10 on Kv4 channels are qualitatively similar
to the effects of DPP6 (235), although, like DPP6, DPP10
also appears to be expressed predominantly in the brain
(235). It also seems unlikely, therefore, that DPP10 plays
a role in the generation of cardiac Kv channels. It is
certainly possible, however, that there are additional
members of this family that are expressed in the myocar-
dium and that remain to be identied and characterized.
C. Molecular Correlates of Cardiac Transient
Outward Kv Channels
All available evidence suggests that Kv -subunits of
the Kv4 subfamily encode rapidly activating, inactivating,
1230 JEANNE M. NERBONNE AND ROBERT S. KASS
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and recovering cardiac transient outward Kv channels
referred to as I
to,f
(Table 1). In rat and mouse ventricular
myocytes exposed to antisense oligodeoxynucleotides
(AsODNs) targeted against Kv4.2 or Kv4.3, for example,
I
to,f
density is reduced by 50% (169, 193). Signicant
reductions in rat ventricular I
to,f
density are also seen in
cells exposed to an adenoviral construct encoding a trun-
cated Kv4.2 subunit (Kv4.2ST) that functions as a domi-
nant negative (238). In addition, it has been reported that
I
to,f
is eliminated in ventricular myocytes isolated from
transgenic mice expressing a pore mutant of Kv4.2,
Kv4.2W362F, that functions as a dominant negative,
Kv4.2DN (43). Taken together, these results demonstrate
that members of the Kv4 subfamily underlie I
to,f
in mouse
and rat ventricles. Biochemical studies have also shown
that Kv4.2 and Kv4.3 are associated in adult mouse ven-
tricles, suggesting that functional mouse ventricular I
to,f
channels reect the heteromeric assembly of the Kv4.2
and Kv4.3 -subunits (193). Given that the properties of
the currents classied as I
to,f
in other species (Table 1)
are very similar to mouse (and rat) I
to,f
, it seems reason-
able to suggest that Kv4 -subunits also underlie I
to,f
in
other species. In dog and human myocardium, however,
Kv4.2 appears not to be expressed (266), suggesting that
only Kv4.3 contributes to I
to,f
in larger mammals. Direct
biochemical and/or molecular evidence to support this
hypothesis, however, has not been provided to date. In
addition, multiple splice variants of Kv4.3 have been iden-
tied in human (387) and rat (490) heart, although the
functional roles of these variants in the generation of
cardiac I
to,f
channels remain to be determined.
It has been demonstrated that KChIP2 coimmunopre-
cipitates with Kv4.2 and Kv4.3 -subunits from adult
mouse ventricles, consistent with a role for this subunit in
the generation of functional Kv4-encoded mouse ventric-
ular I
to,f
channels (193). An important structural role of
KChIP2 in the generation of myocardial I
to,f
channels is
suggested by the observation that I
to,f
is eliminated in
ventricular myocytes isolated from mice with a targeted
disruption of the KChIP2 locus (271). In both canine and
human heart, it has been demonstrated that there is a
gradient in KChIP2 message expression across the thick-
ness of the (left and right) ventricular walls (429, 431),
observations interpreted as suggesting that KChIP2 un-
derlies the observed differences in I
to,f
densities in myo-
cytes isolated from the epicardial, midmyocardial, and
endocardial layers of the (human and canine) ventricles
(429, 431). Although this point remains somewhat contro-
versial (129), it has been reported that KChIP2 protein
expression in canine ventricles parallels KChIP2 message
expression (429), lending further support to the hypothe-
sis that KChIP2, not Kv4.3, underlies the gradient in ca-
nine (and human) ventricular I
to,f
densities. In rat and
mouse, however, there is no detectable gradient in
KChIP2 message or protein expression in the ventricles
(193, 431), and it appears that differences in Kv4.2 expres-
sion underlie the regional variations in I
to,f
densities in
rodents (137, 193). Thus there seem to be two potentially
important differences between I
to,f
in rodents and I
to,f
in
large mammals, including humans. In rat and mouse, I
to,f
channels reect the heteromeric assembly of Kv4.2,
Kv4.3, and KChIP2, and differences in Kv4.2 expression
underlie regional differences in I
to,f
densities. In large
mammals, however, I
to,f
channels appear to be produced
by the coassembly of Kv4.3 and KChIP2, and KChIP2
appears to be the primary determinant of the observed
regional differences in I
to,f
densities.
Although the Kv accessory subunits were originally
identied based on association with Kv1 -subunits and
have been considered to be Kv1 -subunit specic, recent
studies suggest a functional role for Kv1 subunits in the
generation of cardiac I
to,f
channels (12). Electrophysio-
logical studies, for example, have revealed that I
to,f
den-
sities are decreased in ventricular myocytes isolated from
mice bearing a targeted deletion of the KCNAB1 gene,
which encodes Kv1 subunits (12). In addition, biochem-
ical studies revealed that Kv4.2 and Kv4.3 coimmunopre-
cipitate with the Kv1 splice variants, Kv1.1 and Kv1.2,
from adult mouse ventricles (12). Taken together, these
observations suggest that (mouse) ventricular I
to,f
chan-
nels function as multimeric protein complexes compris-
ing the Kv4.2 and Kv4.3 pore-forming -subunits and the
accessory Kv1.1, Kv1.2, and KChIP2 subunits (Fig. 5).
The targeted disruption of Kv1 reduces the cell surface
membrane expression of Kv4 -subunits (12), further sug-
gesting that Kv1 functions to regulate the assembly
and/or the trafcking of mouse ventricular I
to,f
channels
from the endoplasmic reticulum to the cell surface (12).
The Kv1 COOH-terminal core domain has been
shown to interact with NH
2
-terminal tetramerization (T1)
domains in Kv1 -subunits (191), and recent studies sug-
gest that Kv4 -subunit NH
2
-terminal domains structurally
resemble Kv1 T1 domains (451). It seems reasonable to
suggest, therefore, that Kv4 NH
2
termini are likely in-
volved in mediating the interaction with Kv1 subunits.
As noted above, however, it has also previously been
demonstrated that Kv4 -subunit NH
2
-terminal domains
are also important in mediating the interactions with
KChIPs (21, 451). Taken together, these observations sug-
gest that Kv4 NH
2
-terminal domains are multifunctional,
mediating -subunit/-subunit interactions, as well as the
associations with the accessory KChIP2 and Kv1 sub-
units. It has also been reported, however, that Kv1 sub-
units regulate the cell surface expression of Kv4.3 sub-
units in heterologous expression systems through inter-
actions with the COOH, not the NH
2
, terminus (566). It is
not clear if Kv1 subunits play a role in the generation of
I
to,f
(and/or other) channels in large mammals, including
humans, primarily because this possibility has not been
explored directly. Given the heterogeneity of subunits
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that can affect Kv4 channel properties in heterologous
expression systems (130), it seems reasonable to suggest
that additional accessory subunits or regulatory proteins
might be involved in mediating the interaction(s) between
Kv4 and Kv1 subunits. It is certainly also possible that
the interactions between Kv4 and Kv1 subunits are
indirect, mediated, for example, by other accessory sub-
units, such as KChIP2 or KChAP (278) or through scaf-
folding proteins (39) or components of the actin cytoskel-
eton (401, 529). In addition, there could well be further
complexity in the subunit composition of I
to,f
channels, as
well as in I
to,f
channel regulation and posttranslational
processing in some cell types/species. Further studies
focused on dening all of the molecular components of
functional myocardial I
to,f
(and other) channels are
needed to provide insights into the detailed molecular
mechanisms involved in the regulation and modulation of
these channels in the normal and in the diseased myocar-
dium.
Electrophysiological studies on atrial myocytes iso-
lated from Kv4.2DN mice revealed that, similar to the
ndings in ventricular cells (43), I
to,f
is eliminated (563).
There are some differences, however, in the properties of
mouse (and rat) ventricular and atrial I
to,f
(26, 43, 68, 69,
75, 79, 194, 562, 563), differences that may reect varia-
tions in the subunit composition of the channels and/or in
posttranslational processing of these subunits. Further
studies focused on detailing the molecular compositions
and the mechanisms controlling the expression and func-
tioning of I
to,f
channels in other cell types, particularly
atrial, nodal, and Purkinje cells, in rodents and in large
animals, are needed to dene denitively the similarities/
differences in the molecular compositions of I
to,f
channels
in different cell types/species.
The kinetic and pharmacological properties of slow
transient outward myocardial Kv currents, referred to as
I
to,s
(Table 1), are different from I
to,f
, observations inter-
preted as suggesting that the molecular correlates of I
to,s
and I
to,f
channels are also distinct. Direct support for this
hypothesis was provided in studies (196) completed on
myocytes isolated from (Kv1.4 /) mice with a targeted
disruption in the KCNA4 (Kv1.4) locus (305). The wave-
forms of the outward currents in cells isolated from the
ventricles of Kv1.4 / animals are indistinguishable
from those recorded in wild-type ventricular cells (196).
In cells isolated from the interventricular septum of Kv1.4
/ animals, however, I
to,s
is undetectable, thereby dem-
onstrating directly that Kv1.4 underlies I
to,s
(196). Given
the similarities in the time- and voltage-dependent prop-
erties of I
to,s
(Table 1) in other species (77, 83, 178, 294,
545, 546), it seems reasonable to suggest that Kv1.4 also
encodes I
to,s
in ferret, rabbit, canine, and human atrial and
ventricular myocytes.
Interestingly, it has also been reported that I
to,s
and
the Kv1.4 protein are upregulated in the right and left
ventricles of Kv4.2DN-expressing transgenic animals (43,
194, 196), suggesting that electrical remodeling occurs in
the myocardium when I
to,f
is eliminated. When the
Kv4.2DN transgene is expressed in the Kv1.4 / null
background, however, both I
to,f
and I
to,s
are eliminated
and, interestingly, no further electrical remodeling is evi-
dent (194). Indeed, electrophysiological recordings from
Kv4.2DN-expressing Kv1.4 / cells revealed that the
waveforms of the Kv currents in RV, LV, and interventric-
ular septum cells are indistinguishable (194). Although
these observations suggest that the molecular mecha-
nisms underlying the observed electrical remodeling in
Kv4.2DN ventricles is highly specic for Kv1.4, it is pres-
ently unclear which of the many possible transcriptional,
translational, and/or posttranslational mechanisms (430)
might be operative. Future studies focused on delineating
the molecular mechanisms involved in the regulation of
ion channel remodeling in this and other mouse models
will likely provide important new mechanistic insights.
D. Molecular Correlates of Cardiac Delayed
Rectier Kv Channels
As noted above, KCNH2 has been identied as the
locus of mutations underlying one form of familial long
QT syndrome, LQT2 (119). Heterologous expression of
KCNH2 cRNA in Xenopus oocytes reveals voltage-gated,
inwardly rectifying K

-selective channels with properties


similar to cardiac I
Kr
channels (448, 499), observations
interpreted as suggesting that KCNH2 encodes I
Kr
(448).
Subsequent studies identied NH
2
- and COOH-terminal
splice variants of the ERG1 protein (272, 282, 304), and
recent biochemical studies suggest that an NH
2
-terminal
ERG1 splice variant, ERG1b, coassembles with the full-
length ERG1a protein to form heteromeric I
Kr
channels in
rat, human, and canine heart (240). The role of the COOH-
terminal variants of ERG1, ERG1-USO (272) in the gener-
ation of functional cardiac I
Kr
channels, however, is pres-
ently unclear. It has been reported that heterologously
expressed KCNH
2
and minK (KCNE1) coimmunoprecipi-
tate (341) and that antisense oligodeoxynucleotides tar-
geted against minK attenuate I
Kr
amplitudes in AT-1 (an
atrial tumor line) cells (567). It has also been reported
that heterologous coexpression of another member of the
KCNE subfamily of accessory subunits, MiRP1 (KCNE2),
modies the properties of KCNH2-encoded Kv currents
(5). It is presently unclear, however, whether the minK or
MiRP1 (or both) accessory subunits associate with
ERG1a and/or ERG1b in adult human heart and contrib-
ute to the generation of functional cardiac I
Kr
channels.
The availability of specic anti-ERG1 antibodies that can
be exploited to immunoprecipitate ERG1 proteins from
heart (240) should make it possible to explore directly the
association between the minK/MiRP accessory subunits
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and the pore-forming ERG1 subunits and the functional
roles of these interactions in the generation of myocardial
I
Kr
channels.
Biochemical studies have now revealed that the heat
shock proteins, Hsp70 and Hsp90, coimmunoprecipitate
with heterologously expressed ERG1 and that geldanamy-
cin, a specic inhibitor of Hsp90, prevents the maturation
(posttranslational processing) and increases the proteo-
somal degradation of the ERG1 protein (163). Interest-
ingly, the interactions between the ERG1 protein and
Hsp70/Hsp90 are increased in LQT2 trafcking decient
KCNH2 mutants, such as ERG1G601S (180). In addition,
the mutant ERG1G601S protein is retained in the endo-
plasmic reticulum (163). Importantly, it has also been
demonstrated that inhibition of Hsp90 decreases func-
tional I
Kr
densities in isolated ventricular myocytes (163).
Taken together, these results suggest that Hsp70 and
Hsp90 function as chaperone proteins, bringing mature
ERG1 protein complexes to the cell surface to generate
functional I
Kr
channels (163). It is certainly possible that
there are additional components of myocardial I
Kr
chan-
nels that inuence the properties and/or the functional
cell surface expression of these channels, and further
studies are needed to test these hypotheses directly.
Heterologous expression of KCNQ1, the locus of mu-
tations in LQT1 (522), reveals rapidly activating, noninac-
tivating Kv currents, whereas coexpression with KCNE1
(minK) produces slowly activating Kv currents similar to
cardiac I
Ks
(40, 447). These observations, together with
biochemical data demonstrating that heterologously ex-
pressed KvLQT1 and minK proteins associate (447), have
been interpreted as suggesting that minK coassembles
with KvLQT1 to form functional cardiac I
Ks
channels (40,
447). In addition, the nding that mutations in the trans-
membrane domain of minK alter the properties of the
KCNQ-1 encoded Kv channels was interpreted as suggest-
ing that the transmembrane segment of minK contributes
to the channel pore (185, 487, 492, 527). Nevertheless, and
similar to the suggested interaction between ERG1 and
MiRP1 (and/or minK), there is presently no direct bio-
chemical/molecular evidence demonstrating a functional
interaction between the minK and KvLQT1 proteins
and/or that minK/KvLQT1 interactions play a role in the
generation of cardiac I
Ks
channels.
A yeast two-hybrid screen, using the intracellular
cytoplasmic COOH terminus of minK as the bait, led to
the identication of a novel LIM-domain-containing pro-
tein, fh12 (274). Heterologous expression studies further
suggest that fh12 is required for the generation of func-
tional cell surface KvLQT1/minK (I
Ks
) channels (274).
These observations suggest that fh12 is required for the
proper assembly of the KvLQT1 and minK subunits, the
trafcking of assembled channels, and/or the cell surface
expression of functional KvLQT1/minK (I
Ks
) channels.
Nevertheless, direct biochemical evidence for coassembly
of the KvLQT1 protein with minK, with fh12 and/or with
any other Kv channel accessory subunits (Table 5) in the
myocardium has not been provided, and the subunit stoi-
chiometry of functional myocardial I
Ks
channels remains
to be determined. Similar to KCNH
2
, splice variants of
KCNQ1, which exert a dominant negative effect when
coexpressed with the full-length KvLQT1 protein (236),
have also been described, although the roles of these
variants in the generation of I
Ks
channels in vivo remain to
be determined.
A variety of experimental strategies, primarily in
mice, have been exploited in studies focused on dening
the molecular correlates of several of the other types of
cardiac delayed rectier Kv currents (Table 1). A role for
Kv1 -subunits in the generation of mouse ventricular
I
K,slow
, for example, was revealed with the demonstration
that I
K,slow
is selectively attenuated in ventricular myo-
cytes isolated from transgenic mice expressing a trun-
cated Kv1.1 -subunit, Kv1.1N206Tag, that functions as a
dominant negative (303). It was subsequently shown,
however, that I
K,slow
is also reduced in ventricular myo-
cytes expressing a dominant negative mutant of Kv 2.1,
Kv2.1N216 (560). Further analyses revealed that there are
actually two distinct components of wild-type mouse ven-
tricular I
K,slow
: one that is sensitive to micromolar con-
centrations of 4-AP and encoded by Kv1 -subunits, and
another that is sensitive to TEA and encoded by Kv2
-subunits (263, 560, 587). These currents are now re-
ferred to as I
K,slow1
and I
K,slow2
, respectively (263, 291,
587). Subsequent studies revealed that I
K,slow1
is selec-
tively eliminated in ventricular myocytes isolated from
mice in which Kv1.5 has been deleted, suggesting that
Kv1.5 encodes the micromolar 4-AP-sensitive mouse ven-
tricular I
K,slow1
(302). These ndings, together with the
previous results obtained on cells isolated from Kv1.4
/ animals (305), in which I
to,s
is eliminated (196),
suggest that, in contrast to the Kv4 -subunits (193), the
Kv1 -subunits, Kv1.4 and Kv1.5, do not associate in adult
mouse ventricles in situ. Rather, functional Kv1 -subunit-
encoded Kv channels in mouse ventricular myocytes ap-
pear to be homomeric, composed of Kv1.4 -subunits
(I
to,s
) or Kv1.5 -subunits (I
K,slow1
). The roles of Kv chan-
nel accessory subunits in the generation of these myocar-
dial Kv1 -subunit-encoded Kv channels, however, remain
to be dened.
Electrophysiological studies completed on isolated
rat atrial myocytes (74, 75), and later on canine (577),
human (19, 531), and mouse (69) atrial myocytes, demon-
strated the presence of a novel component of delayed
rectication, referred to as I
Kur
(I
Kultrarapid
), with time-
and voltage-dependent properties that are quite distinct
from I
Kr
and I
Ks
(479). Although I
Kur
appears to be an
atrial specic current in large mammals, the properties of
I
K,slow1
in mouse ventricular myocytes are indistinguish-
able from (rat, human, and canine) atrial I
Kur
(168, 562,
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586). Human, rat, and canine atrial I
Kur
, like mouse ven-
tricular I
K,slow1
, activates rapidly and undergoes little or
no inactivation during brief depolarizations, properties
similar to those seen on heterologous expression of sev-
eral different Kv -subunits, including Kv1.2, Kv1.5, Kv3.1,
and others. In addition, I
Kur
, like I
K,slow1
, is sensitive to
micromolar concentrations of 4-AP (168, 562, 586). The
later nding led to the hypothesis that Kv1.5 likely en-
codes human and rat atrial I
Kur
(75, 533). Direct experi-
mental support for this hypothesis was provided with the
demonstration that exposure to antisense oligode-
oxynucleotides targeted against Kv1.5 selectively attenu-
ates I
Kur
in isolated adult human (159) and rat (68) atrial
myocytes. The important physiological role for Kv1.5 in
human atria is suggested by the nding that I
Kur
densities
and Kv1.5 protein expression are reduced markedly in the
atria of patients with chronic atrial brillation (511).
Although it was reported that Kv3.1, rather than
Kv1.5, functions in canine atria to encode I
Kur
(479),
subsequent work demonstrated that Kv3.1 is not detect-
able in canine atria, whereas Kv1.5 (message and protein)
is robustly expressed (157). It seems reasonable to con-
clude, therefore, that, similar to other Kv channels, the
molecular correlate of cardiac I
Kur
(Kv1.5) is similar
across species. At present, it is unclear if Kv accessory
subunits play a role in the generation of atrial (mouse, rat,
canine, or human) I
Kur
. Unexpectedly, however, it has
now been demonstrated that Kv1 subunits do not asso-
ciate with Kv1.5 in adult mouse ventricles and that the
targeted deletion of Kv1 has no detectable effect on
mouse ventricular I
Kur
(I
K,slow1
) (12). It may well be,
however, that other Kv channel accessory subunits con-
tribute to the generation of I
Kur
channels. Further studies,
focused on dening the molecular composition of I
Kur
channels and the roles of accessory subunits, are needed
to dene the underlying molecular mechanisms involved
in the regulation of I
Kur
channels in the normal and dis-
eased myocardium.
VII. MOLECULAR COMPONENTS OF OTHER
CARDIAC POTASSIUM CHANNELS
A. Inwardly Rectifying Cardiac K

(Kir) Channel
Pore-Forming -Subunits
Similar to the Kv channels, functionally distinct types
of myocardial inwardly rectifying K

channels (378) are


formed by the association of diverse inward rectier K

(Kir) channel pore-forming -subunit genes (140). Several


Kir subunit subfamilies, Kir1 through Kir6, most with
several members, have been identied (Table 6) and, like
Kv -subunits, Kir -subunits also assemble as tetramers
to form functional K

selective channels (Fig. 5). Also


similar to Kv channel -subunits, a unifying terminology
has been developed for naming the Kir -subunit proteins
(Kir1.xKir6.x) and the genes (KCNJ1KCNJ15) encod-
ing these proteins (199). In contrast to the Kv -subunits,
however, the Kir -subunits have two (not six) transmem-
brane domains (Fig. 5).
Based on the properties of heterologously expressed
Kir subunits, it was suggested that -subunits of the Kir2
subfamily likely encode the strongly inwardly rectifying
Kir channels, I
K1
, in cardiac cells (140, 378), and all
(three) members of the Kir2 subfamily (Table 6) are ex-
pressed in the myocardium (298, 488). Interestingly, the
KCNJ2 gene, which encodes Kir2.1, has been identied as
the locus of mutations in Andersens syndrome (243, 403),
an inherited disorder that is often life-threatening owing
to QT prolongation and cardiac (ventricular) arrhythmias.
Similar to Timothys syndrome (479), however, Ander-
sens syndrome is actually a multisystem disorder involv-
ing the cardiovascular, skeletomuscular, and other sys-
tems, and typically, Andersens syndrome patients
present initially with developmental abnormalities (494).
The mutations in KCNJ2 that are associated with Ander-
sens syndrome that have been described to date appear
to result in mutant Kir2.1 proteins that function in a
dominant negative fashion to suppress Kv2.x-encoded I
K1
currents (11, 280, 409). Individuals carrying Andersens
syndrome mutations in KCNJ2 can display QT prolonga-
tion (Long QT7), periodic paralysis, as well as craniofacial
malformations (11, 22, 494, 498), alone or in combination.
Because only the KCNJ2 gene appears to be affected in
Andersens syndrome, the multisystem nature of this dis-
order likely reects the fact that Kir2.x-encoded channels
are expressed and are functional in a variety of cells/
tissues. Myocardial I
K1
(and other K

channels) have been


shown to be regulated directly by phosphatidylinositol
bisphosphate (PIP
2
) (209, 219, 470, 489). Interestingly,
many of the Andersens mutations are in the PIP
2
binding
region of Kir2.1 (139), suggesting that the regulation/mod-
ulation of I
K1
channels by PIP
2
is altered and that it is the
alterations in the modulatory effects of PIP
2
that underlie
the phenotypic consequences of the Andersens syndrome
mutations.
The rst direct molecular evidence that Kir2 -sub-
units encode cardiac I
K1
channels was provided in studies
completed on myocytes isolated from mice bearing a
targeted disruption of the coding region of Kir2.1 (Kir2.1
/) or Kir 2.2 (Kir2.2 /) (580, 581). Although the
Kir2.1 / mice have cleft palate and die shortly after
birth, thereby precluding electrophysiological studies on
adult cells (580), experiments completed on isolated new-
born Kir2.1 / ventricular myocytes revealed that I
K1
is
absent (581). Interestingly, however, an inwardly rectify-
ing current, with properties distinct from the wild-type
I
K1
, is evident in Kir2.1 / myocytes (581), suggesting
either that an additional Kir current component is
present, but difcult to resolve in wild-type cells in the
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presence of I
K1
or, alternatively, that a novel I
K1
is up-
regulated in Kir2.1 / hearts. In contrast to the ndings
in Kir2.1 / cells, voltage-clamp recordings from adult
Kir2.2 / ventricular myocytes revealed that I
K1
densi-
ties are reduced, compared with I
K1
densities in wild-type
cells, and that the properties of the residual I
K1
currents
(in Kir2.2 / cells) are indistinguishable from wild-type
I
K1
(581). These results were interpreted as suggesting
that both Kir2.1 and Kir2.2 contribute to (mouse) ventric-
ular I
K1
channels, and subsequent studies provided bio-
chemical and molecular evidence to support this hypoth-
esis (342, 592). The observation that the inwardly rectify-
ing channels remaining in the absence of Kir2.1 have
properties distinct from the endogenous I
K1
channels fur-
ther suggests that functional cardiac I
K1
channels are
heteromeric. Consistent with this hypothesis, detailed
comparisons of the properties of heterologously ex-
pressed Kir2.1, Kir2.2, and Kir2.3 -subunits and endoge-
nous guinea pig and sheep atrial and ventricular myocytes
suggests marked regional and cell type specic differ-
ences in the molecular composition of I
K1
channels (133).
Further studies focused on dening the molecular com-
positions of myocardial I
K1
channels in different cell types
and in different species, including humans, are needed to
dene the molecular diversity and the functioning of these
channels.
In the heart, weakly inwardly rectifying I
KATP
chan-
nels are thought to play a role in both myocardial isch-
emia and preconditioning (232, 377, 380). In heterologous
systems, I
KATP
channels can be reconstituted by coex-
pression of Kir6.x subunits with ATP-binding cassette
proteins that encode the sulfonylurea receptors, SURx
(31, 456). Although previous pharmacological and molec-
ular studies suggest that cardiac sarcolemmal I
KATP
chan-
nels reect the heteromeric assembly of Kir6.2 and
SUR2A subunits, Kir6.1 is also expressed in the heart
(406), and exposure of isolated (rat neonatal) ventricular
myocytes to antisense oligodeoxynucleotides against
TABLE 6. Diversity of inwardly rectifying K

(Kir) and two-pore domain K

(K2P) channel -subunits


Family Subfamily Protein Gene
Location
Cardiac
Current Family Subfamily Protein Gene
Location
Cardiac
Current Human Mouse Human Mouse
Kir1 K2P
Kir1 TWIK
Kir1.1 KCNJ1 11q25 9A4 ?? TWIK-1 KCNK1 1q42 8E2 ??
TWIK-2 KCNK6 19q11 ??
Kir2
Kir2.1 KCNJ2 17q23 11E1 I
K1
TWIK-3 KCNK7 11q12 19A
TWIK-4 KCNK8 11q12
Kir2.2 KCNJ12 17p11.2 11B1.3 I
K1
TREK
Kir2.3 KCNJ4 22U ?? TREK-1 KCNK2 1q41 1H6 I
ss
??
TREK-2 KCNK10 14q32
Kir2.4 KCNJ14 19q13.4 7B3 ?? TASK
Kir3 TASK-1 KCNK3 2p24 ??
Kir3.1 KCNJ3 2C1.1 I
KACh
TASK-2 KCNK5 6p21.1 14A1
Kir3.2 KCNJ6 21q22 16C4 TASK-3 KCNK9 8q24.3
KCNJ7 TASK-4 KCNK14
Kir3.3 KCNJ9 1q21 1H2.3 TASK-5 KCNK15 20q12
TRAAK
Kir3.4 KCNJ5 11q25 9A4 I
KACh
TRAAK-1 KCNK-4 11q12 19A
Kir4 THIK
Kir4.1 KCNJ10 1q21 1H2.3 THIK-1 KCNK13 14q32 12E
Kir4.2 KCNJ15 21q22 16C4 THIK-2 KCNK12 2p21 ??
Kir5 TALK
Kir5.1 KCNJ16 17q25
Kir6 TALK-1 KCNK16 6p21
Kir6.1 KCNJ8 12p11.1 6G2 ?? TALK-2 KCNK17 6p21 ??
Kir6.2 KCNJ11 11p15 783 I
KATP
Boxes denote cardiac expression.
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SUR1 reduces I
KATP
channel densities (572). These obser-
vations suggest the interesting possibility that there may
be some molecular heterogeneity among cardiac I
KATP
channels. The absolute requirement for the Kir6.2 subunit
in the generation of cardiac I
KATP
channels, however, was
unequivocally demonstrated in studies completed on
mice in which the Kir6.2 gene was disrupted by homolo-
gous recombination (292, 456, 484). Voltage-clamp record-
ings from ventricular myocytes isolated from these
(Kir6.2 /) animals revealed no detectable I
KATP
chan-
nel activity (292, 484). These ndings clearly suggest that
Kir6.1 alone (i.e., in the absence of Kir6.2) cannot gener-
ate functional myocardial I
KATP
channels. Nevertheless, it
is certainly still possible that Kir6.1 coassembles with
Kir6.2 to form Kir6.1/Kir 6.2 heteromeric cardiac I
KATP
channels.
The suggestion that SUR2 plays a pivotal role in the
generation of cardiac I
KATP
channels is supported by the
nding that I
KATP
channel density is reduced in myocytes
from animals (SUR2 /) in which SUR2 has been de-
leted (411). In contrast, there are no measurable cardiac
effects of the targeted disruption of SUR1 (455, 456).
Nevertheless, it is interesting to note that I
KATP
channel
density is reduced, i.e., the channels are not eliminated, in
SUR2 /ventricular myocytes, and the properties of the
residual I
KATP
channels in SUR2 / ventricular myo-
cytes are similar to those of the channels produced on
heterologous coexpression of Kir6.2 and SUR1 (411).
These ndings strongly suggest that SUR1 likely also
coassembles with Kir 6.2 in ventricular myocytes to pro-
duce functional I
KATP
channels (at least in the absence of
SUR2). Immunohistochemical studies suggest that Kir6.2
and SUR2A assemble to form plasmalemmal cardiac I
KATP
channels, whereas Kir6.1, Kir6.2 and SUR2A are ex-
pressed in mitochondria, observations interpreted as sug-
gesting that the molecular compositions of functional I
Kr
channels in different cellular compartments are distinct
(473). Similar to I
K1
channels, myocardial I
KATP
channels
are also modulated by the binding of PIP
2
and other
membrane lipids (209, 470).
The waveforms of action potentials recorded from
isolated Kir6.2 / ventricular myocytes are indistin-
guishable from those recorded from wild-type cells (484).
These observations clearly suggest that I
KATP
channels do
not play a role in shaping action potential waveforms (in
mouse ventricles) under normal physiological conditions.
The action potential shortening typically observed in
wild-type ventricular cells during ischemia or metabolic
blockade, however, is abolished in Kir6.2 / ventricular
cells (484). In addition, the protective effect of ischemic
preconditioning is abolished in Kir6.2 / hearts (456,
485), and infarct size in Kir6.2 / animals, with and
without preconditioning, is the same (485). These obser-
vations are consistent with the hypothesis that cardiac
I
KATP
channels play an important role under pathophysi-
ological conditions, particularly those involving metabolic
stress (74, 577). Interestingly, however, it has been dem-
onstrated that action potential durations are also largely
unaffected in transgenic animals expressing mutant I
KATP
channels with markedly (40-fold) reduced ATP sensitivity
(268). The mutant I
KATP
channels would be expected to be
open (owing to the reduced sensitivity to closure by ATP)
at rest and to markedly affect cardiac membrane excit-
ability. The fact that action potentials are unaffected in
ventricular myocytes expressing mutant I
KATP
channels
clearly suggests that additional inhibitory regulatory
mechanisms play a role in the physiological control of
cardiac I
KATP
channel activity in vivo (268). Further stud-
ies focused on dening and characterizing these regula-
tory mechanisms will be of considerable interest.
B. Two-Pore Domain K

(K2P) Channel
Pore-Forming -Subunits
In addition to the many Kv (Table 4) and Kir (Table 6)
channel -subunits, a novel type of K

pore-forming
-subunit with four transmembrane spanning regions and
two pore domains (K2P) was identied with the cloning of
TWI
K
-1 (289), now referred to as KCNK1 (199). Studies in
heterologous systems suggest that functional K2P chan-
nels, unlike Kv and Kir channels that assemble as tetra-
mers, reect the dimeric assembly of (two) K2P -sub-
units and that each of the (two) pore domains in each
-subunit contributes to the formation of the K

-selective
pore (286). Subsequent to the identication of TWI
K
-1, a
rather large number of K2P -subunit genes, KCNK1
KCNK17, were identied, and a subset of these appears to
be expressed in the myocardium (Table 6). Similar to the
Kv and Kir channels, a systematic terminology has been
developed for naming the (KCNK) genes encoding K2P
-subunits.
Heterologous expression studies have demonstrated
that the members of various K2P subunit subfamilies give
rise to K

-selective currents with distinct time- and volt-


age-dependent properties and differential sensitivities to
a variety of modulators, including pH, fatty acids, and
anesthetics (286, 287). It seems likely, therefore, that K2P
subunit-encoded K

channels could be important in reg-


ulating the normal physiological functioning of the adult
mammalian heart, as well, perhaps, as inuencing myo-
cardial responses to pathophysiological stimuli. Direct
experimental support for this hypothesis was provided
with the demonstration that the pathophysiological ef-
fects of platelet activating factor on the myocardium are
directly linked to inhibition of KCNK3 (TASK-1)-encoded
(or closely related) K

channels in ventricular myocytes


(39). Interestingly, the effect of platelet activating factor is
dependent on protein kinase C (39), although the under-
lying molecular mechanisms have not been detailed. Al-
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though it has been demonstrated that different KCNK-
encoded K2P -subunits coassemble to form heteromeric
channels (55), it is presently unclear whether heteromeric
K2P -subunit assembly is physiologically relevant in the
myocardium. Similarly, there is very little information
presently available about the role(s) of accessory sub-
units and/or other regulatory molecules in the generation
of K2P -subunit-encoded myocardial channels.
The facts that there are so many K2P -subunits
(Table 6), that many of them are ubiquitously expressed,
and that the properties of K2P -subunit-encoded chan-
nels are regulated by a variety of potentially relevant
physiological (and pathophysiological) stimuli suggest
that channels encoded by K2P -subunits likely subserve
a variety of important physiological functions. Experi-
mental support for this hypothesis was provided with the
demonstration that mice bearing a targeted disruption of
the KCNK2 gene (which encodes TREK-1, Table 6) dis-
play increased sensitivity to epilepsy and ischemia (208).
It is unclear, however, whether there is a cardiac pheno-
type in the KCNK2 / mice, primarily because this
possibility appears not to have been addressed (208). The
physiological roles of TREK-1 and of each of the other
K2P channel -subunits expressed in the myocardium, as
well as in other cell types, therefore, remain largely un-
known. Both TREK-1 and TASK-1 are expressed in the
heart, and heterologous expression of either of these
subunits alone gives rise to instantaneous, noninactivat-
ing K

currents that display little or no voltage depen-


dence (286). These observations have led to suggestions
that these subunits contribute to myocardial back-
ground or leak K

currents (32), although presently,


there is no direct experimental evidence to support this
hypothesis. Interestingly, however, the properties of the
currents produced on expression of TREK-1 or TASK-1
are similar to those of the current referred to as I
Kp
identied in guinea pig ventricular myocytes (576), as well
as to I
ss
in mouse ventricular myocytes (79, 562). Further
studies focused on dening the roles of each of the K2P
-subunits in the generation of myocardial K

channels,
and the roles of these channels in the physiological, as
well as the pathophysiological, functioning of the heart,
are needed to clarify these issues.
VIII. MYOCARDIAL POTASSIUM CHANNELS
AND THE ACTIN CYTOSKELETON
Similar to myocardial Nav channels, considerable ev-
idence has now accumulated to suggest that several dif-
ferent types of plasmalemmal K

channels in cardiac cells


also interact with components of the actin cytoskeleton
and that these interactions play important roles in regu-
lating the properties, the trafcking, and/or the anchoring
of these channels. It has been shown, for example, that
myocardial I
KATP
channels likely are linked to, and regu-
lated by, the actin cytoskeleton (179, 336). Exposure to
cytochalasin D, which disrupts/destabilizes actin la-
ments, for example, accelerates the rundown of cardiac
I
KATP
channels, whereas actin lament stabilizers inhibit
channel rundown (179). It appears that cytochalasin D
exerts its effects by interfering with the interaction be-
tween SUR2A and Kir6.2 subunits, thereby modifying
SUR-mediated regulation of the I
KATP
channels (76, 571).
These observations suggest that the biophysical proper-
ties, as well as the cell surface expression of I
KATP
chan-
nels, are affected by cytoskeletal interactions. Similarly,
the biophysical properties, including rectication and
Ca
2
(but not Mg
2
) sensitivity, of myocardial I
K1
chan-
nels are affected by treatment with cytochalasin D (336).
There is also experimental evidence suggesting that
the functioning of Kv channels in myocardial (and other)
cells is regulated and/or modulated through interactions
with the actin cytoskeleton. It has been demonstrated, for
example, that exposure to phalloidin, which stabilizes
actin laments, markedly reduces action potential dura-
tions, whereas treatment with cytochalasin D (or cytocha-
lasin B) prolongs action potential durations, in hypertro-
phied rat ventricular myocytes (568). Voltage-clamp stud-
ies revealed that the cytochalasin- and phalloidin-
mediated effects on action potentials reect the specic
attenuation or augmentation, respectively, of I
to,f
(568).
These observations suggest that functional cardiac Kv4
-subunit-encoded I
to,f
channels are regulated/modulated
directly or indirectly through interactions with the ac-
tin cytoskeleton. Interestingly, experiments in heterol-
ogous expression systems also suggest that the modu-
lation of Kv1.5, which encodes cardiac I
Kur
channels, by
protein kinases and phosphatases, requires an intact
cytoskeleton (333).
Similar to cardiac Nav channels, the interactions be-
tween functional myocardial Kir and Kv channels and the
actin cytoskeleton are assumed to be mediated through
association with actin-binding proteins and/or other scaf-
folding proteins, suggesting that Kir and Kv channels also
function as multimeric protein complexes (Fig. 6). Con-
sistent with this hypothesis, it was recently demonstrated
that Kir2.x -subunits associate with several scaffolding
proteins, including CASK, veli-3, mint-1, and SAP-97 (285).
Interestingly, Kir subunits in brain also interact with a
very large number and variety of PDZ domain-containing
proteins (285, 372). In addition, it has been reported that
Kir6.x subunits interact directly with the 143-3 protein
and that this interaction is requisite for the functional cell
surface expression of assembled Kir6.x-SUR-encoded
I
KATP
channels (575). It has also been reported that Kv
-subunits in several subfamilies bind to PDZ-containing
proteins, including PSD-95 and SAP-97 (145, 237, 557,
558), although the physiological signicance of these ob-
servations in terms of the expression and/or the function-
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ing of cardiac Kv channels has not been determined. The
interactions between Kv1 and Kv4 -subunits and PSD-95
impact the recruitment of Kv1- and Kv4-encoded channels
into lipid rafts (558). This nding may explain early ob-
servations suggesting that the expression of Kv4 -sub-
units alone fails to reveal targeting to lipid rafts (327).
More importantly, these observations suggest that spe-
cic associations between Kv -subunits and PDZ-con-
taining scaffolding proteins play an important role in the
targeting of functional Kv channels to specic subcellular
domains. The targeting of Kv channels to specic subcel-
lular compartments would be expected to have rather
profound effects on the regulation of membrane excitabil-
ity and conduction through the myocardium (270, 463). In
addition, Kv channel targeting could facilitate specic
interactions between Kv channels and modulatory/regu-
latory proteins, including protein kinases and phospha-
tases, as has been clearly demonstrated in the protein
kinase A-mediated regulation of cardiac I
Ks
channels, that
appears to be mediated by an A-kinase anchoring protein
or AKAP (330, 331).
Interestingly, it has also been demonstrated that Kv1
-subunits contain PDZ-binding domains (145) and that
Kv1.5 binds directly to the actin-binding protein -acti-
nin-2 (329). Further studies revealed that members of
three subfamilies of Kv -subunits, Kv1.5, Kv2.1, and
Kv4.2, bind to -actinin-2, interactions that affect the
properties and the functional expression of the resulting
Kv -subunit-encoded K

currents (329). These observa-


tions suggest that several Kv -encoded Kv channels
likely also interact directly with the actin cytoskeleton via
-actinin-2 (Fig. 6). It has also been reported that Kv4
-subunits interact directly with lamin (401) and that
this interaction regulates the functional cell surface ex-
pression and the localization of Kv4-encoded channels
(401). Subsequent studies revealed that actin depolymer-
ization modulates the cell surface expression of heterolo-
gously expressed Kv4-encoded channels (529). Similar to
Nav channels, these observations suggest the interesting
and potentially important hypothesis that myocardial Kv
channels also function as components of macromolecular
complexes containing the channel components and a va-
riety of scaffolding and regulatory proteins linked to the
actin cytoskeleton (Fig. 6).
Recently, it was also suggested that Kir3-encoded
channels interact with integrin (344), suggesting that myo-
cardial K

channel expression and functioning may also


be linked to the extracellular matrix. Although clearly in
the very early stages, it seems reasonable to suggest that
the link between the cytoskeleton (as well, perhaps, as
the extracellular matrix) in the regulation of myocardial
K

channel expression, localization, and functioning has


been made. Further studies, aimed at exploring the roles
of the cytoskeleton and the extracellular matrix in regu-
lating myocardial K

channel expression, distribution,


and functioning, as well as those focused on probing the
underlying molecular mechanisms, will likely provide im-
portant new insights into the physiological and patho-
physiological roles of these interactions.
IX. SUMMARY AND CONCLUSIONS
Electrophysiological studies have identied multiple
types of voltage-gated inward and outward currents ex-
pressed in cardiac cells (Table 1). The outward K

cur-
rents are more numerous and more diverse than the in-
ward (Na

and Ca
2
) currents, and most cardiac myo-
cytes express multiple voltage-gated, as well as inwardly
rectifying, K

channels (Table 1). These (K

) channels
are the primary determinants of myocardial action poten-
tial repolarization, and regional differences in K

channel
densities and properties underlie observed variations in
action potential waveforms and contribute to the genera-
tion of normal cardiac rhythms. Voltage-gated inward
Ca
2
channel currents and the Na

channel window
current, however, also contribute to myocardial action
potential repolarization. The pivotal role played by the
Nav channel window current, for example, has been
elegantly demonstrated in electrophysiological studies
characterizing mutations in SCN5A that underlie long
QT3, as well as in computer-based simulations (Fig. 4) of
cellular electrical activity (73, 90, 141, 188).
Molecular cloning has revealed an unexpected diver-
sity of ion channel pore-forming -subunits (Tables 2, 4,
and 6) and accessory subunits (Tables 3 and 5) that
contribute to the formation of the various inward and
outward current-carrying channels (Table 1) identied
electrophysiologically in myocardial cells. Similar to the
electrophysiological diversity of myocardial K

channels
(Table 1), the molecular analysis has revealed that multi-
ple voltage-gated (Kv) (Table 4) and inwardly rectifying
(Kir) (Table 6) K

channel pore-forming -subunits, as


well as a number of accessory subunits of these (Kir and
Kv) channels (Table 5), are expressed in the myocardium.
A variety of in vitro and in vivo experimental approaches
have been exploited to probe the relationship(s) between
these subunits and functional myocardial K

channels,
and important insights have been provided through mo-
lecular genetics and the application of techniques that
allow functional channel expression to be manipulated
directly. In contrast to the progress made in dening the
roles of the various Kv and the Kir -subunits in the
generation of functional myocardial Kv and Kir channels,
there is very little known about the functional roles of the
K2P -subunits (Table 6). In addition, the specic and/or
multiple functional roles of most of the known K

chan-
nel accessory subunits (Table 5) remain to be claried.
Dening the molecular correlates/compositions of the
various myocardial K

channels will facilitate future ef-


1238 JEANNE M. NERBONNE AND ROBERT S. KASS
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forts focused on delineating the molecular mechanisms
controlling the properties and the functional expression
of these channels.
In addition to the diversity of pore-forming and ac-
cessory channel subunits (Tables 26), several recent
studies, exploiting a combination of molecular, biochem-
ical, and electrophysiological approaches, have revealed a
rather staggering array of proteins that seem likely to
contribute to regulating the properties, the cell surface
expression, and/or the subcellular localization of func-
tional myocardial membrane ion channels (Fig. 6). Taken
together, the results of these studies suggest that myocar-
dial ion channels function as macromolecular protein
complexes. Interestingly, several components of these
macromolecular protein channel complexes provide links
to the actin cytoskeleton and the extracellular matrix
(Fig. 6), suggesting important functional links between
different ion channel complexes in cardiac cells, as well
as between myocardial structure and electrical function-
ing. Understanding this molecular complexity clearly de-
mands that novel experimental approaches, such as pro-
teomics and genomics, be exploited to identify the vari-
ous components of functional ion channel complexes, the
sites of protein-protein interactions, and the underlying
mechanisms involved in mediating these interactions.
These areas represent important areas for further re-
search focus in efforts directed towards dening the de-
tailed mechanisms involved in the regulation/modulation
of myocardial membrane excitability and the generation
of normal cardiac rhythms.
Numerous studies have documented changes in func-
tional ion channel expression during normal cardiac de-
velopment, as well as in damaged or diseased heart. It has
also been demonstrated that electrical remodeling occurs
in the heart in response to changes in electrical activity or
cardiac output, and this remodeling is directly attributed
to changes in the functional expression and/or the prop-
erties of the various ion channels that underlie myocardial
action potential generation. Although there are numerous
possible (transcriptional, translational, and posttransla-
tional) mechanisms that could be involved in regulating
the functional expression and the properties of these
channels, very little is presently known about the under-
lying molecular mechanisms that are important in medi-
ating the changes in channel expression evident during
normal development, as well as in conjunction with myo-
cardial damage, disease, and/or electrical remodeling. An
important focus of future research efforts will almost
certainly be on exploring these mechanisms in detail.
ACKNOWLEDGMENTS
We thank past and present members of our laboratories for
their contributions to the understanding of the molecular basis
of cardiac repolarization. In addition, we are indebted to Rick
Wilson for his expert assistance with the generation of the tables
and gures presented in this review and to Dr. Kevin Sampson
for conducting the simulations illustrated in Figures 4 and 7.
Address for reprint requests and other correspondence:
J. M. Nerbonne, Dept. of Molecular Biology and Pharmacology,
Washington University Medical School, 660 South Euclid Ave.,
St. Louis, MO 63110 (E-mail: jnerbonne@msnotes.wustl.edu).
GRANTS
We gratefully acknowledge the long-standing and contin-
ued nancial support for our research endeavors provided by
the National Heart, Lung, and Blood Institute of the National
Institutes of Health.
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doi:10.1152/physrev.00002.2005 85:1205-1253, 2005. Physiol Rev
Jeanne M. Nerbonne and Robert S. Kass
Molecular Physiology of Cardiac Repolarization
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