CHAPTER

SODIUM AND POTASSIUM:

CHANNELS AND PUMPS

INTRODUCTION: TRANSPORT ACROSS MEMBRANES

9
Before examining the important roles of the alkali metals sodium and potassium, we should briefly
review how ions are transported across membranes. As we pointed out in Chapter 3, Structural and
Molecular Biology for Chemists, the phospholipid bilayer of biological membranes is essentially
impermeable to polar molecules and to ions
the permeabilities of Na1 and K1 are of the order of
10212 cm21
the corresponding value for H2O, is around 1022. So, simple diffusion would not suf-
fice to explain the millisecond transmission of nerve impulses. Transport across membranes is con-
ferred by two classes of membrane proteins, namely, channels and pumps. Channels allow ions to
flow down a concentration gradient by a process known as passive transport or facilitated diffusion.
Of course, channels cannot remain open all of the time, and so they are usually gated, which simply
means that, like regular garden gates, they usually remain shut, and can only be opened, either by the
binding of a ligand (ligand-gated), or by changes in the membrane potential (voltage-gated), which
administers the ‘push’ required to open the gate. Ligand-gated channels, like the acetylcholine recep-
tors in postsynaptic membranes are opened by the binding of the neurotransmitter acetylcholine to its
membrane-bound receptor, whereas the voltage-gated sodium and potassium channels, which mediate
the action potentials in neuronal axons described below, are opened by membrane depolarization.
In contrast, pumps use energy, either in the form of ATP or light, to drive the unfavourable
uphill transport of ions or molecules against a concentration gradient; in other words, they are
involved in active transport. There are two types of ATP-driven pumps, so-called P-type ATPases
and ABC (ATP-binding cassette) transporters, both of which use conformational changes induced
by ATP binding and its subsequent hydrolysis to transport ions across the membrane. The
Na1-K1-ATPase (NKA) described below is one of the P-type ATPases, which achieves uphill
exchange of cytoplasmic Na1 ions for extracellular K1 ions using ATP-mediated phosphorylation,
followed by autodephosphorylation, to drive conformational changes that allow access to the bind-
ing sites of the pump from only one side of the membrane at a time. Another mechanism of active
transport, which uses the electrochemical gradient of one ion to drive the counter-transport of
another will be illustrated by the Na1/H1 exchanger, crucial among other things for the control of
intracellular pH. Yet another example, discussed in Chapter 11, Calcium: Cellular Signalling, is the
Na1/Ca21 exchanger, which plays an important role in removing Ca21 from cells.

SODIUM VERSUS POTASSIUM

Sodium and potassium are relatively abundant in the earth’s crust, although sodium is much more
prevalent in sea water. The Na1 and K1 contents in the average man represent about 1.4 g/kg and

Biological Inorganic Chemistry. DOI: http://dx.doi.org/10.1016/B978-0-12-811741-5.00009-6

© 2019 Elsevier B.V. All rights reserved. 261
262 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

2.0 g/kg, respectively, making them among the most important of metal ions in terms of concentra-
tion. However, their distribution is quite different. Whereas in most mammalian cells, 98% of K1
is intracellular, for Na1 the situation is the reverse. This concentration differential ensures a num-
ber of major biological processes, such as cellular osmotic balance, signal transduction and neuro-
transmission. It is maintained by the NKA, which we will discuss below. However, despite the
presence of only 2% of total body K1 outside of cells, this extracellular K1 concentration plays a
crucial role in maintaining the cellular membrane resting potential. Fluxes of these alkali metal
ions play a crucial role in the transmission of nervous impulses both within the brain and from the
brain to other parts of the body. The opening and closing of gated ion channels (gated channels),
which are closed in the resting state, and which open in response to changes in membrane potential,
generates electrochemical gradients across the plasma membranes of neurons. A nerve impulse is
constituted by a wave of transient depolarization/repolarization of membranes which traverses the
nerve cell, and is designated an action potential. Hodgkin and Huxley (1952) demonstrated that a
microelectrode implanted into an axon (the long process emanating from the body of a nerve cell)
can record an action potential (Fig. 9.1A). In the first B0.5 ms the membrane potential increases
from around 260 mV to about 130 mV, followed by a rapid repolarization, which overshoots the
resting potential (hyperpolarization) before slowly recovering. The action potential results from a
rapid and transient increase in Na1 permeability followed by a more prolonged increase in K1 per-
meability (Fig. 9.1A). The opening and closing of these gated Na1 and K1 ion channels across the
axonal membranes creates the action potentials (essentially electrochemical gradients) across these
membranes, which allows information transfer and also regulates cellular function.
The regulation of the flow of ions across cell membranes is absolutely essential for the function-
ing of living cells. Because of the hydrophobicity of cellular membranes (as we saw earlier), the
energetically driven preference of ionic species like Na1 K1, Cl2, H1 and Ca21 to cross, never
mind to find themselves preferentially on one side or other of a biological membrane, would be
impossible. Without ionic gradients, which maintain high concentrations of K1 and low concentra-
tions of Na1 within the cell, cells would not be able to carry out their normal metabolic activities.
This means, in simplistic terms, that some molecular machines must be able to distinguish between
Na1 and K1 ions (presumably unhydrated, since the degree of hydration could make for difficulties
in discrimination). So, before even beginning a discussion of ‘active’ transport proteins, whether
ion pumps or ion exchangers, we ask the question how do potential transporters distinguish between
these two closely related cations?
Over the last 50 years of research on synthetic and naturally occurring small molecules which
bind ions (host/guest chemistry with ions), basic rules of ion selectivity have been established with
small molecules. Two major factors are important
the atomic composition and the stereochemis-
try (in particular, the size) of the binding site. Using synthetic chemistry, molecules have been cre-
ated of a given class with selectivity favouring Li1 (radius 0.60 Å), Na1 (radius 0.95 Å), K1
(radius 1.33 Å) and Rb1 (radius 1.48 Å) by simply adjusting the cavity size to match the ion
(Dietrich, 1985).1
Now that we have high-resolution crystal structures of membrane transport proteins, including
K1 and, more recently Na1, channels, we can begin to understand how ion selectivity is
accomplished.

1
The author remarked that this was particularly easy for the alkali metals of group 1A because the differences in ionic
radius are greatest (0.35 Å).
 POTASSIUM CHANNELS 263

Na+ equilibrium potential

(A) +60

+40

Membrane potential (mV)

+20 Action potential

–20
Depolarization
–40
Resting potential
–60
Hyperpolarization

–80 K+ equilibrium potential

(B) 30
Ionic permeabilities (mmho • cm–2)

Na+ permeability
20

10

K+ permeability

0
0 1 2 3 4
Time (ms)

FIGURE 9.1
Time course of an action potential.
From Voet, D., Voet, J.G., 2004. Biochemistry, third ed. John Wiley and Sons, Hoboken, pp. 1591.
Reproduced with permission from John Wiley and Sons, Inc.

POTASSIUM CHANNELS
K1 channels selectively transport K1 across membranes, hyperpolarize cells, set membrane poten-
tials and control the duration of action potentials, among a myriad of other functions. They use
diverse forms of gating, but they all have very similar ion permeabilities. All K1 channels show a
selectivity sequence of K1 B Rb1 . Cs1, whereas the transport of the smallest alkali metal ions
Na1 and Li1 is very slow
typically the permeability for K1 is at least 104 that of Na1. The
determination of the X-ray structure of the K1 ion channel has allowed us to understand how it
264 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

S1 S2 S3 S4 S5 Pore loop S6
N C

+ ++ +
EAGSENSFFKSIPDAFWWAVVTMTTVGYGDMTPVGVWGK
***** * * ** **

OUT

IN
C C
N

FIGURE 9.2
One of the first pictures of a tetrameric K1 channel with a selectivity filter made of pore loops. A linear
representation of a Shaker K1 channel subunit on top shows shaded hydrophobic segments S1 to S6 and a region
designated the pore loop. A partial amino acid sequence from the Shaker K1 channel pore loop highlights amino
acids shown to interact with extracellular scorpion toxins (¬), intracellular tetraethylammonium (m), and K1 ions
(1). The pore loop was proposed to reach into the membrane (middle) and form a selectivity filter at the centre of
four subunits (bottom).
From MacKinnon, R., 2004. Potassium channels and the atomic basis of selective ion conduction (Nobel Lecture). Angew. Chem.
Int. Edn. 43, 4265
4277. Copyright 2004 with permission from John Wiley and Sons.

selectively filters completely dehydrated K1 ions, but not the smaller Na1 ions. Not only does this
molecular filter select the ions to be transported, but also the electrostatic repulsion between K1
ions, which pass through this molecular filter in Indian file, provides the force to drive the K1 ions
rapidly through the channel at a rate of 107
108 per second (reviewed in Doyle et al., 1998;
MacKinnon, 2003, 2004).2
The first voltage-gated potassium channel to be identified was the gene encoding the Shaker
mutation3 in the fruit fly Drosophila. Fig. 9.2 presents the first pictures of the tetrameric Shaker
K1 channel with a selectivity filter made of pore loops. A linear representation of a Shaker K1
channel subunit on top shows shaded hydrophobic segments S1 to S6 and a region designated the
pore loop. A partial amino acid sequence from the Shaker K1 channel pore loop highlights amino

2
The 2003 Nobel Prize for Chemistry was awarded to Rod MacKinnon for his pioneering work in this area.
3
These mutant fruit flies shake violently when anaesthetized with ether.
 POTASSIUM CHANNELS 265

acids shown to interact with the extracellular inhibitor scorpion toxin (¬), the intracellular tetra-
ethylammonium (m) and K1 ions (1). The pore loop was proposed to reach into the membrane
(middle) and form a selectivity filter at the centre of four subunits (bottom).
The first structure determination of a K1 channel was the bacterial K1 channel KcsA from
Streptomyces lividans, which has a simple topology with only two membrane-spanning segments
per subunit, corresponding to the Shaker K1 channel without the S1
S4 segments. Currently, we
have the structures of a number of K1 channels
pH-dependent bacterial K1 channels, voltage-
gated and calcium-gated K1 channels from bacteria and voltage-gated mammalian K1 channels.
What is most striking is that they all have a similar architecture. They are all tetramers with four-
fold symmetry about the central K1-conducting pore. On the basis of hydrophobicity analysis, there
are two closely related families of K1 channels, those containing two membrane-spanning seg-
ments per subunit, like KcsA and those containing six, like the Drosophila and the vertebrate
voltage-gated K1 channels. In the latter case, the last two transmembrane helices, S5 and S6,
together with the P-loop which connects them, constitute the pore itself. Several other families of
ion channels have similar architectures, including the eukaryotic voltage-gated Na1 channels. The
two membrane-spanning families include the inwardly rectifying K1 channels, and some bacterial
K1 channels. They are made up of four subunits, each having only two transmembrane segments.
The analogous M1 and M2 segments and pore loop, form the complete transmembrane structure
of the two transmembrane K1 channels. Sequence homology is very high between the two families
in the channel region, particularly in the pore region itself. K1 channels allow some other monova-
lent cations through (but not Na1), do not allow the passage of anions and are blocked by divalent
cations.
The Kþ channel pore is comprised of four usually identical subunits, which encircle a central
ion conduction pathway with fourfold symmetry (Fig. 9.3). The KcsA K1 channel has what is often
referred to as an ‘inverted tepee’4 structural arrangement, some 45 Å long, and made up of three
distinct regions of variable width. Two of the four subunits are shown for the KcsA K1 channel in
Fig. 9.3B. Each subunit contains two fully transmembrane α-helices termed inner (nearest the ion
pathway) and outer (nearest the membrane) and a tilted pore helix (red in Fig. 9.4A), which runs
half way through the membrane, pointing its C-terminal negative end-charge towards the ion path-
way. Near the midpoint of the membrane, the ion pathway is very wide, forming a central water-
filled cavity nearly 10 Å in diameter, with a hydrated K1 ion at its centre (Zhou et al., 2001). In
order to catalyse high conduction rates, the channel must overcome the electrostatic repulsion that
a K1 ion would normally experience when moving from bulk water into the low dielectric mem-
brane environment. By allowing the K1 ion to remain hydrated at the membrane centre, and by
directing the C-terminal negative end-charge of α-helices towards the ion pathway, a K1 ion is sta-
bilized at the centre of the membrane (Roux and MacKinnon, 1999).
In the extracellular third of the ion pathway, between the central cavity and the extracellular
solution (Fig. 9.4A, gold), is the selectivity filter, some 0.3 nm wide and 1.2 nm in length, contain-
ing a highly conserved TVGYG amino acid sequence, the characteristic K1 channel signature
found in K1 channels throughout all living organisms. By attaching monoclonal Fab fragments to
KcsA, it was possible to obtain a high-resolution structure of the KcsA channel at a resolution
of 2.0 Å, instead of the 3.2 Å resolution of the KcsA protein alone (Zhou et al., 2001). From the

4
The wigwam of American Indians.
266 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

(A)

Out

In

(B)

Out Pore helix

Outer
helix

Inner
helix N
In
bundle
Inner helix

FIGURE 9.3
(A) A ribbon representation of the KcsA K1 channel with its four subunits coloured differently. The channel is
oriented with the extracellular solution on top.
From MacKinnon, R., 2004. Potassium channels and the atomic basis of selective ion conduction (Nobel Lecture). Angew. Chem.
Int. Edn. 43, 4265
4277. Copyright 2004 with permission from John Wiley and Sons.

2.0-Å resolution structure, it is clear that the selectivity filter consists of four unhydrated K1 bind-
ing sites. The filter consists of four evenly spaced layers of carbonyl oxygen atoms and a single
layer of threonine hydroxyl oxygen atoms, which create four K1 ion binding sites numbered 1 to 4
from the extracellular to the intracellular side (Fig. 9.4B). Each of these sites can bind a dehydrated
 (A) (B)

(C)

= +

1,3 2,4
(D)

1,3 2,4
configuration configuration

FIGURE 9.4
The ion conduction pore of K1 channels. (A) Two of the four subunits from the KcsA pore are shown with the
extracellular side on top. Each subunit contains an outer helix close to the membrane, an inner helix close to the
pore, a pore helix (red) and a selectivity filter (gold). Blue mesh shows electron density for K1 ions and water
along the pore. (B) Close-up view of the selectivity filter with dehydrated K1 ions at positions 1 through 4
(external to internal) inside the filter and a hydrated K1 ion in the central cavity below the filter. (C) Electron
density in the filter corresponds to two configurations of K1 ions (green) alternating with water molecules (red):
1,3 contains K1 at positions 1 and 3, 2,4 contains K1 at positions 2 and 4. (D) Throughput cycle for K1
conduction invoking 1,3 and 2,4 configurations.
From MacKinnon, R., 2003. Potassium channels. FEBS Lett. 555, 62
65.
Copyright 2003 with permission from John Wiley and Sons.
268 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

K1 ion, which interacts with eight carbonyl oxygens (main chain carbonyl or side-chain hydroxyl
atoms) of the TVGYG amino acid sequence. Mutations in this sequence lead to disruption of the
ability of the channel to distinguish between K1 ions and Na1 ions. The K1 ions are bound in an
essentially dehydrated state, surrounded by eight oxygen atoms from the protein, four ‘above’ and
four ‘below’ each ion (Fig. 9.4B). This arrangement of protein oxygen atoms surrounding each
binding site in the selectivity filter is very similar to the arrangement of water molecules around
the hydrated K1 ion observed in the central cavity. The high-resolution crystal structure provides
an elegant demonstration of a row of K1 binding sites that mimic the waters of hydration surround-
ing a K1 ion. Potassium ions are therefore able to diffuse from water into the selectivity filter
where the energetic cost of dehydration is compensated. Sodium ions, on the other hand, do not
seem to enter the selectivity filter in crystal structures even when Na1 is present in vast excess

reduction of K1 to 3 mM in the presence of 150 mM Na1 causes the selectivity filter to undergo a
conformational change to a ‘collapsed’ state. From the relative electron density of the sites, it is
clear that each site is occupied by K1 only half of the time, in other words that at any given time
only two of the binding sites are occupied, with water molecules in the intermediate sites. Thus
two K1 ions permanently occupy the sites 1 and 3 or 2 and 4, with a water molecule sandwiched
between them (Fig. 9.5). The X-ray structures also give support to a previously suggested ‘knock-
on’ mechanism whereby K1 ions can traverse the channel. Additional K1 ions coordinated by eight
water molecules are observed at the extracellular mouth of the channel and in the central cavity.
When one of these ions enters either end of the filter, it displaces the equilibrium of the two K1
ions already resident, with the consequence that the column of K1 ions moves along until one of
the ions is ejected, and the new K1 ion takes its place in the filter. The 1,3 and 2,4 configurations
are the endpoints of the conduction cycle shown in Fig. 9.4D. This crystallographic model is in
excellent agreement with Hodgkin and Keynes, who proposed that the K1 conduction mechanism
involves the single file movement of two to three ions across the membrane (Hodgkin and Keynes,
1955).
We saw earlier how the 3-D structure of the two transmembrane bacterial K1 channels, KcsA,
analogous to the inwardly rectifying K1 channels, reveals an ‘inverted tepee’ arrangement around a
central pore, with the narrow outer mouth of the pore formed by the pore loop. KcsA was crystal-
lized under conditions which favour its closed conformation (Doyle et al., 1998). Information on
how the pore might be opened comes from the structure of a bacterial 2TM calcium-activated K1
channel, MthK, analysed in its calcium-bound, open form (Jiang et al., 2002). The structures of
KcsA and MthK are shown in Fig. 9.5. The major structural difference between these two channels
is the position of their inner helices on the intracellular side of the selectivity filter. In KcsA, the
inner helices are straight and form a helix bundle near the intracellular membrane surface. At the
bundle crossing in KcsA, the pore narrows to about 3.5 Å in diameter and is lined with hydropho-
bic amino acids, creating a barrier to the flow of K1 ions. In contrast, the MthK inner helices are
bent at a highly conserved glycine residue, and this bend appears to function as a hinge, opening
the intracellular mouth of the pore sufficiently to allow permeation of ions and splayed open so
that the central cavity becomes confluent with the cytoplasm, leaving free access for ion flow
between the cytoplasm and the selectivity filter.
Finally, we might ask just how does the membrane voltage trigger opening or closing of the
channel? It has been proposed that when a voltage-gated channel opens, charged amino acids
(called gating charges) move through the electric field of the membrane, coupling electrical work
to the opening process. For a voltage-gated K1 channel, the gating charge is equivalent to almost
 POTASSIUM CHANNELS 269

Inner Helix
Filter

Closed Opened

FIGURE 9.5
Closed and opened conformations of the pore. Sequences of ligand- and voltage-gated K1 channels are from
Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, J., Chait, B.T., et al., 2002. The open pore conformation of
potassium channels. Nature 417, 523
526. The selectivity filter sequence is highlighted orange and the glycine
gating hinge red. Three subunits of the closed pore conformation of KcsA (left) and the opened pore
conformation of MthK (right) are shown with the selectivity filter and gating hinge coloured to match the
sequence highlights.
From MacKinnon, R., 2003. Potassium channels. FEBS Lett. 555, 62
65.
Copyright 2003 with permission from John Wiley and Sons.

14 electrons moving all the way across the transmembrane voltage difference. This can be attrib-
uted to four arginine residues per subunit (16 in total). The pore of the voltage-gated K1 KvAP
channel is surrounded by α-helical voltage sensors (Fig. 9.5A) (Jiang et al., 2003), which are bound
to monoclonal Fab fragments in the high-resolution structure (Fig. 9.6A). The gating charge argi-
nine residues are located on hydrophobic helix-turn-helix structures, which have been designated as
voltage sensor paddles (Fig. 9.6B), since it was suggested that they could move within the mem-
brane at the protein
lipid interface when the channel opens. This was tested in lipid membranes by
tethering biotin to various positions and assessing whether avidin5 can access the biotin from either

5
Avidin, a protein rich in egg white, binds ‘avidly’ to the B vitamin biotin, and is often used in biological applications to
detect biotin-labelled proteins.
270 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

(A)

(C)

External S3b

S3b
S4
(B)
30Å

External

S4

FIGURE 9.6
The voltage-gated K1 channel KvAP. (A) A tetramer from the crystal viewed down the fourfold axis from the
cytoplasm (Jiang et al., 2003). The channel is α-helical with four subunits shown in different colours. Monoclonal
Fab fragments (green) used to crystallize KvAP are attached to the voltage sensors. (B) A single subunit of KvAP
is shown with the extracellular side on top. Helical elements S3b and S4 form a hydrophobic ‘voltage sensor
paddle’ with gating charge arginine residues. In the crystal, the Fab is attached to the voltage sensor paddle and
has extended it towards the cytoplasm. (C) CPK model of the voltage sensor paddle shown in approximate closed
(left) and opened (right) depths relative to the 30 Å hydrophobic core. Tethered biotin (red and yellow) attached
to position 121 is accessible to internal avidin when the channel is closed and to external avidin when the channel
is opened. Avidin engulfs the red segment when it binds to biotin. A and B are from Jiang et al., 2003.
From MacKinnon, R., 2003. Potassium channels. FEBS Lett. 555, 62
65.
Copyright 2003 with permission from John Wiley and Sons.

side of the membrane (Jiang et al., 2003). At certain positions on one face of the paddle, tethered
biotin is dragged all the way across the membrane from the intracellular side in the closed confor-
mation to the extracellular side in the opened conformation (Fig. 9.6C). This implies that biotin is
accessible to avidin from the intracellular side when the channel is closed and from the
 SODIUM CHANNELS 271

extracellular side when it is opened, involving movements large enough to transfer the four arginine
gating charges most of the way across the membrane (MacKinnon, 2003).

SODIUM CHANNELS
Whereas the structures of homotetrameric K1 channels have been available for over two decades,
the structures of eukaryotic NaV and Cav channels have resisted determination to date. This is
essentially due to the fact that the eukaryotic Nav and Cav channels lack the structural fourfold
symmetry of Kv channels. Eukaryotic Nav channels consist of a pore-forming α-subunit, which has
undergone extensive posttranslational modifications, associated with auxiliary β-subunits (β-1-4).
Although the α-subunit alone is sufficient for functional expression of the channel properties, the
kinetics and voltage dependence of channel gating are modified by the β-subunits. The α-subunits
are single polypeptide chains of B2000 residues, organized in four homologous domains (I
IV),
each consisting of six transmembrane α-helices (S1
S6) and an additional P-loop located between
the S5 and S6 helices (Fig. 9.7A). Each domain is similar to the individual subunits of the K1
channels described above. The four domains assemble to form a pseudotetramer, with the S5 heli-
ces, intervening P-regions, and S6 helices comprising the pore subdomain that forms the pathway
for the translocation of ions across the cell membrane. The S1
S4 helices form the voltage sensor
subdomains.
In the last 5
6 years, it has been found that, in contrast to eukaryotic NaV channels, bacterial
Nav (BacNav) channels are biochemically more tractable, can be readily crystallized and their struc-
tures give molecular details of the basic design principles which are found in the more complex
eukaryotic Nav channels (for reviews, see Corry and Thomas, 2012; Catterall and Zheng, 2015;
Ahern et al., 2015; Naylor et al., 2016; Clairfeuille et al., 2017). The sequence of BacNav is analo-
gous to one domain of the eukaryotic NaV channel (Fig. 9.7B), and functions, like its eukaryotic
equivalent, as a tetramer, but made up of four identical subunits. The first structure of a BacNav
channel, from Arcobacter butzleri (NavAb) (Payandeh et al., 2011), revealed four identical subunits
packed symmetrically to form a functional channel with a central ion-conducting pore (Fig. 9.8A,B).
The four S5 and S6 helices and the P loops constitute the pore, surrounded by the S1
S4 helices
which form the voltage-sensing module (VSM) (Fig. 9.8C). The overall topography is similar to that
of the K1 channel, in the form of an inverted tepee, but differs significantly from the K1 channel, as
we will see in the ‘selectivity filter,’ which is both wider and shorter as well as being lined by amino
acid side chains.
The NavAb pore consists of an external vestibule, a narrow selectivity filter, a spacious water-
filled central cavity and an activation gate at the inner end of the S6 helix (Fig. 9.8D), which is in
the closed conformation. The helix-loop-helix motif formed by the P1 and P2-helices forms the
scaffold of the selectivity filter (Fig. 9.8C). The P2-helix lines an electronegative extracellular ves-
tibule that attracts cations towards the pore. The characteristic signature sequence of the selectivity
filter, TLESWS (Fig. 9.9), is located between the P1 and P2-helices, forming a B5 Å wide ion con-
duction pathway. Four Glu side-chains (TLESWS) directly line the entrance to the filter, followed
by two concentric rings of backbone carbonyls (TLESWS) which promote the passage of hydrated
sodium ions (Fig. 9.9C). In contrast to the constrained, dehydrating selectivity filters found in K1
272 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

(A) (B)
Nav channel NaChBac
channel
I II III IV Outside
cell

Pore N

Voltage
sensing Inactivation C

N
Inside
cell

FIGURE 9.7
Voltage-gated sodium channel (NaV) structure. (A) 2D schematic map of NaV structure and function. The α subunit
of NaV1.2 is illustrated as a transmembrane folding diagram in which cylinders represent transmembrane α-helices
and lines represent connecting amino acid sequences in proportion to their length. The roman numerals indicate the
four homologous domains and the arabic numerals are used to label the six transmembrane helices. The S4 helices
are coloured in red with ‘1’ signs indicating gating charges. The S5
S6 helices are coloured in green and the small
white circles indicate key residues in the selectivity filter with ‘1’ and ‘
’ signs indicating their charge states. The
yellow circle with an ‘h’ indicates the inactivation gate. (B) Schematic map of the bacterial NaChBac channel, which
contains the minimal functional elements of a single homologous domain in a mammalian NaV.
From Catterall, W.A., Zheng, N., 2015. Deciphering voltage-gated Na(1) and Ca(21) channels by studying prokaryotic ancestors.
Trends Biochem. Sci. 40, 526
534. Copyright 2015 with permission from Elsevier.

channels, ion conduction appears to occur through a mechanism which prefers passing hydrated
Na1 ions to passing hydrated K1 or Ca21 ions. Analogous to eukaryotic Nav channels (Heinemann
et al., 1992), BacNav channels can be converted into highly selective Ca21 channels through simple
mutation of the selectivity filter sequence (e.g. TLDDWSN in the case of NavAb). Recent crystal
structures of this so-called CavAb channel have captured hydrated Ca21 ions bound in a multi-ion
pore configuration (Fig. 9.9D) (Tang et al., 2014).

THE SODIUM
POTASSIUM ATPASE (NKA)

Mammalian cells maintain a lower concentration of Na1 (around 12 mM) and a higher concentra-
tion of K1 (around 140 mM) than in the surrounding extracellular medium (145 mM and 4 mM,
 THE SODIUM
POTASSIUM ATPASE (NKA) 273

(A) (B)
VSM
VSM PM VSM

VSM
PM
S5

S6
S4-S5 Linker
VSM VSM Intracellular

(C) P2 (D)
Voltage-sensor domain
P2 External
Selecitivity vestibule
filter
S2 S1
P1 P Selectivity
S4
filter
S6 S5

S5 Central
cavity

S3 Activation
S6
Pore domain gate
S4-S5 linker

FIGURE 9.8
Overall structures of prokaryotic voltage-gated sodium channels (NaVs). (A) Structure of the Arcobacter butzleri
bacterial NaV (NaVAb) determined at 2.7-Å resolution and viewed from the extracellular side. The four subunits
of the homotetrameric channel are shown in different colours. The central pore is surrounded by four voltage-
sensing modules (VSMs). (B) Side view of NaVAb with the same colouring scheme as shown in (A). The S5 and
S6 helices of one subunit (slate) and the VSM of another subunit (cyan) are omitted for clarity. (C) View of a
single NavAb subunit colored to highlight different structural regions in BacNav channels (PDB 3RVZ). (D)
Architecture of the NaVAb pore with pore volume shown in grey and the high-strength-field (HSF) site residue
Glu177 shown in sticks. P and P2 indicate the P and P2 helices.
From Catterall, W.A., Zheng, N., 2015. Deciphering voltage-gated Na(1) and Ca(21) channels by studying prokaryotic ancestors.
Trends Biochem. Sci. 40, 526
534. Copyright 2015 with permission from Elsevier.

respectively). The high intracellular K1 and low intracellular Na1 concentrations are maintained
by the NKA which is localized in the plasma membrane. NKA was the first P-type ion translocat-
ing adenosine triphosphatase ever identified (Skou, 1957),6 and is the most prominent member of
the large superfamily of P-type ATPases (for a review, see Palmgren and Nissen, 2011). Other

6
Jens Skou received the 1997 Nobel Prize for Chemistry for this discovery.
274 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

(A) (B)
P2 Extracellular
funnel

P1
Selectivity
filter

S5
Central
cavity

S6
Activation
gate
Side-view

(C) NavMs: TLESWS (D) CavAb: TLDDWSN

N

S S D S

W W
E
D

*L *L

*T *T
Na+ selective Ca+ selective

FIGURE 9.9
Pore module and selectivity filter in BacNav channels. (A) Ribbon rendering of the NavAb pore (PDB 3 RVZ)
with the selectivity filter residue side-chain from Glu177 shown in purple stick representation; two subunits are
omitted for clarity. An approximate pore volume is shown in grey surface as calculated by the program MOLE.
(B) Sectioned view of the NavAb pore (PDB 3RVZ) with electrostatic potential coloured from 210 to 10 kT (red
to blue). The closed S6 activation gate can be appreciated in this view. (C) Selectivity filter of the Na1-selective
NavMs channel (PDB 5BZB). Two subunits are omitted for clarity and residues within the filter are indicated.  T
and  L denote the backbone carbonyls of these residues which line the ion conduction pathway. Presumed Na1
ions are rendered as purple spheres. (D) Selectivity filter of the Ca1-selective CavAb channel (PDB 4Ms2). Ca21
ions are rendered as cyan spheres, where anomalous diffraction was used to verify Ca21 ion binding at these sites.
Based on coordination considerations alone, ions bound within the NavMs or CavAb selectivity filters appear to
be at least partially, if not fully, hydrated.
From Clairfeuille, T., Xu, H., Koth, C.M., Payandeh J., 2017. Voltage-gated sodium channels viewed through a structural biology lens.
Curr. Opin. Struct. Biol. 45, 74
84. Copyright 2017 with permission from Elsevier.

members of the family in eukaryotes are the sarcoplasmic reticulum Ca21 ATPases (SERCA),
gastric H1-K1-ATPases and, in plants, the H1-ATPases.
The overall reaction catalysed by NKA is:
3Na1 ðinÞ 1 2K1 ðoutÞ 1 ATP 1 H2 O ,5. 3Na1 ðoutÞ 1 2K1 ðinÞ 1 ADP 1 Pi :
 THE SODIUM
POTASSIUM ATPASE (NKA) 275

This results in the extrusion of three positive charges for every two which enter the cell, result-
ing in a transmembrane potential of 50 2 70 mV, and has enormous physiological significance.
More than one-third of the ATP utilized by resting mammalian cells is used to maintain the intra-
cellular Na1
K1 gradient (in nerve cells this can rise to up to 70%), which controls cell volume,
allows neurons and muscle cells to be electrically excitable, and also drives the active transport of
sugars and of amino acids.
The reaction cycle of P-type ATPases has been studied extensively, and can be explained by the
E1/E2 theory, according to which the ATPase can exist in two distinct conformational states, E1
and E2. In the light of the vast amount of structural information concerning reaction intermediates
in the sarcoplasmic reticulum Ca21-ATPase (SERCA) mechanism, we will delay a detailed discus-
sion of this subject until Chapter 11, Calcium: Cellular Signalling. The NKA achieves the thermo-
dynamically unfavourable uphill exchange of cytoplasmic Na1 ions for extracellular K1 ions by
using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational
changes that allow ion access to the binding sites of the pump from only one side of the membrane
at a time. How this is achieved is still not entirely clear, but a number of experimental observations
can be put together to provide a plausible mechanism. Key among these is that the ATPase is phos-
phorylated by ATP in the presence of Na1, and that the resulting aspartyl phosphate residue is only
dephosphorylated in the presence of K1. This immediately suggests, as outlined in Fig. 9.10 (inset),
that the enzyme exists in two distinct conformations, E1 and E2, which differ not only in their con-
formation, but in their catalytic activity and their ligand specificity. Indeed, this cation exchange is
accomplished by conformational changes linked to phosphorylation and dephosphorylation of the
pump, which permit strictly alternating access to its binding sites. This is from the extracellular
side in the phosphorylated state in the E2-P state, but from the cytoplasm in the dephosphorylated
state (E1). The E1 form faces towards the inside of the plasma membrane, has a high-affinity site
for Na1, and reacts with ATP to form the ‘high-energy’ aspartyl phosphate intermediate
E1BP.3Na1. In relaxing to its ‘low-energy’ conformation E2-P, the bound Na1 is released. The
outward-facing E2-P, which has a high affinity for K1, binds 2K1, and the aspartyl phosphate
group is hydrolysed to give E2.2K1. This form then binds ATP, changes conformation to the E1
form, releasing its 2K1 inside the cell, thereby allowing the cycle to recommence. The outcome of
this is to couple ATP hydrolysis with the vectorial transport of Na1 and K1 across the plasma
membrane. The inhibition of the NKA by cardiac glycosides like digitalis (an extract of foxglove
leaves), which blocks the dephosphorylation of the E2-P form of the enzyme, is the basis for a num-
ber of steroid drugs which are commonly prescribed for the treatment of congestive heart failure.
The structures of a number of P-type ATPases have been determined (Bublitz et al., 2010;
Toyoshima and Cornelius, 2013). The structure of the SERCA, which is the simplest of the
PII-type ATPases, composed of a single polypeptide chain of 994 residues, has been determined by
far the most frequently. Structures are available for almost all of the intermediates of the reaction
cycle of SERCA. Chapter 11, Calcium: Cellular Signalling, gives a detailed discussion of the mech-
anism of P-type ATPases.
Structural studies on NKA have, in contrast, lagged far behind, not least because NKA is much
more complex. It is made up of the catalytic α-subunit (similar to SERCA, around 1000 residues
long), a β-subunit of about 300 residues, and a tissue-specific regulatory subunit of 702180 residues,
known as FXYD protein. In humans, there are four isoforms of the a-subunit, three of the β-subunit
and seven of FXYD, resulting in a considerable variety of different NKAs. The crystal structure
(Shinoda et al., 2009; Toyoshima et al., 2011) reveals that the catalytic α-subunit (Fig. 9.10), like
276 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

FIGURE 9.10
Architecture of Na1-K1-ATPase from shark rectal gland with bound MgF422 and K1, a stable analogue of the
E2  Pi  2K1 state. (A) A ribbon diagram of NKA with ouabain (shown in space fill) bound at low affinity (PDB
ID: 3A3Y). Colour changes gradually from the N-terminal (blue) to the C-terminal (red). ATP is taken from the
E2(TG)  ATP crystal structure of Ca21-ATPase (SERCA1a) (PDB ID: 3AR4) and docked in the corresponding
position. Bound K1 ions are marked (I, II, and C) and circled. Inset shows a simplified diagram of the E1/E2
scheme. CLR, cholesterol; OBN, ouabain. (B) Digestion sites (proteinase K, black ‘x’; chymotrypsin, magenta ‘x’)
and interaction sites with other proteins (dotted circles). Residue numbers in parentheses refer to corresponding
residues in SERCA1a. AP-2, adaptor protein 2; IP3R, inositol 1,4,5-triphosphate receptor; PI3K, phophoinositide-3
kinase. (C) Disposition of transmembrane helices viewed approximately perpendicular to the membrane from the
cytoplasmic side (NKA, yellow; SERCA1a, cyan). CLR, cholesterol.
From Toyoshima, C., Kanai, R., Cornelius, F., 2011. First crystal structures of Na1, K1-ATPase: new light on the oldest ion pump.
Structure 19, 1732
1738. Copyright 2011 with permission from Elsevier.
 ACTIVE TRANSPORT DRIVEN BY NA 1 GRADIENTS 277

SERCA, is made up of ten α-helices which make up the ion transport domain and three cytoplasmic
domains, the N-domain (nucleotide binding), P-domain (phosphorylation) and A-domain (actuator),
which confer the ATP hydrolysing activity. The N-domain positions the γ-phosphoryl of ATP for
nucleophilic attack, a conserved Asp in the P-domain accepts the phosphoryl group and forms a
high-energy aspartyl-phosphate, while a Glu residue in the A-domain positions a water molecule for
subsequent hydrolysis, which leads to release of the phosphoryl group.

ACTIVE TRANSPORT DRIVEN BY Na 1 GRADIENTS

Many transmembrane transporter proteins, termed secondary transporters, use the discharge of an
ionic gradient to power the ‘uphill’ translocation of a solute molecule across membranes. Coupling
solute movement to ion transport enables these secondary transporters to concentrate solutes by a
factor of 106 with a solute flux 105 faster than by simple diffusion. Sugars and amino acids can be
transported into cells by Na1-dependent symports. Dietary glucose is concentrated in the epithelial
cells of the small intestine by a Na1-dependent symport, and is then transported out of the cells
into the circulation by a passive glucose uniport situated on the capillary side of the cell
(Fig. 9.11). For this system to continue functioning, ATP hydrolysis, which maintains the intracel-
lular Na1 concentration through the NKA, is absolutely required.
Glutamate transporters, also referred to as excitatory amino acid transporters (EAATs), clear
synaptically released glutamate from the extracellular space, thus ensuring the precise control of

Intestinal lumen Capillaries

Na+-glucose symport
Glucose
Glucose uniport

Glucose Glucose

Na+ Na+ Na+

ATP

K+ K+
ADP + Pi

(Na+ – K+)-ATPase
Brush border cell

FIGURE 9.11
Epithelial brush border cells of the small intestine concentrate glucose from the intestinal lumen in symport with
Na1: this is driven by the Na1-K1-ATPase located on the capillary side of the cell. The glucose is then exported
out of the brush border cell by a passive uniport system.
From Voet, D., Voet, J.G., 2004. Biochemistry, third ed. John Wiley and Sons, Hoboken, pp. 1591.
Reproduced with permission from John Wiley & Sons, Inc.
278 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

(A) (B)
OUT 1K+ 1Glu–, 3Na+, 1H+ Cl– 1Glu–, 3Na+, 1H+

T1K+ T T1Glu–, 3Na+, 1H+

(6) (1)

Transporter Glutamate
reorientation (5) (2) 1K+
translocation

(4) (3)
T1K+ T1Glu–, 3Na+, 1H+
T Cell

IN 1K+ 1Glu–, 3Na+, 1H+

FIGURE 9.12
Transport cycle and stoichiometry of EAATs. (A) Simplified state diagram of the EAAT transport cycle. After
glutamate and coupled ions (step 1) bind to the transporter (T), they are translocated (step 2) and released into the
cell cytosol (step 3). Next K1 binds from the intracellular side (step 4) and reorients the substrate-free transporter
(step 5). K1 is released outside the cell (step 6). (B) Cartoon illustrating the ion coupling stoichiometry of an
EAAT. The uptake of 1 glutamate molecule is coupled with the influx of 3Na1, 1H1 and the efflux of 1K1. In
addition, EAATs have a glutamate-activated anion conductance, which results in the influx of chloride under
physiological conditions.
From Jiang, J., Amara, S.G., 2011. New views of glutamate transporter structure and function: advances and challenges.
Neuropharmacology 60, 172
181. Copyright 2011 with permission from Elsevier.

excitatory synaptic transmission. In addition, excessive extracellular concentrations of glutamate can

be neurotoxic and the efficient removal of glutamate limits pathological conditions, associated with
excitotoxic cell death. The transport cycle and stoichiometry of EAATs, of which five mammalian
isoforms have been characterized, are presented in Fig. 9.12. After glutamate and coupled ions bind
to the transporter (1), they are translocated (2) and released into the cell cytosol (3). Next K1 binds
from the intracellular side (4) and reorients the substrate-free transporter (5), and finally K1 is
released outside the cell (6). The ion coupling stoichiometry is shown in Fig. 9.12B. Uptake of one
Glu molecule is accompanied with the influx of 3Na1, 1H1 and the efflux of 1K1. Since EAATs
have Glu-associated anion conductance, in physiological conditions, this results in the influx of Cl2.
Secondary active transporters, including EAATs, are thought to function through an alternating
access mechanism, in which the substrate-binding site is alternatively accessible from the extracel-
lular and intracellular sides through a process that depends on one or more conformational changes.
The determination of a number of crystal structures of the archaeal glutamate transporter ortholo-
gue GltPh from Pyrococcus horikoshii has enabled a general picture to be formed of how EAATs
transport glutamate. GltPh exists as a trimer comprised of three identical subunits in the crystal.
Each protomer contains eight TM domains and two reentrant loops (Fig. 9.13) (Yernool et al.,
2004). The first six TM domains form a scaffold surrounding a C-terminal core domain that con-
tains structural elements required for the transport mechanisms. This C-terminal translocation core
domain includes two opposite-facing helical hairpins (HP1 and HP2), which have been proposed to
 ACTIVE TRANSPORT DRIVEN BY NA 1 GRADIENTS 279

GltPh

Out

In

FIGURE 9.13
Structural features of GltPh. (top). The topology of GltPh. as revealed by the crystallization of an archaeal EAAT
orthologue from Pyrococcus horikoshii, GltPh. (Left) Cartoon representation of the GltPh protomer viewed in the
plane of the membrane. Transmembrane domain s1-6 coloured in grey form a scaffold that holds the core
translocation domain comprised of HP1 (yellow), TM7 (orange), HP2 (red) and TM8 (magenta). Residue A307 in
GltPh is shown as red dots in the topology model and is also illustrated as a blue sphere in the GltPh protomer.
(Right) View of a GltPh trimer parallel to the membrane. Subunits in the trimer are coloured in either green,
magenta or cyan. The protomer and the trimer structure are based on the GltPh crystal structure 1XFH.
From Jiang, J., Amara, S.G., 2011. New views of glutamate transporter structure and function: advances and challenges.
Neuropharmacology 60, 172
181. Copyright 2011 with permission from Elsevier.

act as the inner and outer doors of the transporter, a seventh TM helix interrupted by a β-linker and
an amphipathic helix, TM8, in the crystal. The first GltPh structure to be determined (PDB 1D
1XFH) represents an occluded state, in which both inner and outer gates are closed (Fig. 9.14A).
Two other structures have been determined. A second structure (PDB ID 2NWW), referred to as
the TBOA-bound conformation, was crystallized in the presence of the competitive inhibitor,
DL-threo-benzyloxyaspartate (TBOA) (Boudker et al., 2007). The overall structural features of the
TBOA-bound GltPh are very similar to those of the substrate-bound carrier (PDB ID 1XFH) except
that the HP2 region is displaced away from the substrate-binding site (Fig. 9.14B). A third GltPh
280 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

(A) (B) (C)

TBOA-bound conformation Substrate-bound conformation Inward-facing conformation

(D) (E)

Out Out

In In

Outward facing Inward facing

FIGURE 9.14
Mechanisms of substrate transport. (A) The TBOA-bound GltPh structure (PDB ID 2NWW) showing HP2
displaced from the substrate-binding site (TBOA is removed from the structure for clarity). (B) The substrate-
bound GltPh structure (PDB ID 2NWX) showing the closed HP2 with two sodium ions and the bound substrate,
l-aspartate. (C) The inward-facing GltPh structure (PDB ID 3KBC) in which the whole C-terminal domain moves
towards the cytoplasm. (D) Surface representation of the outward-facing GltPh before the core domain moves into
the cytoplasm. (E) Surface representation of the inward-facing GltPh. The three protomers are coloured in either
green, magenta or cyan. The illustrations are based the GltPh crystal structures 2NWW, 2NWX and 3KBC.
From Jiang, J., Amara, S.G., 2011. New views of glutamate transporter structure and function: advances and challenges.
Neuropharmacology 60, 172
181. Copyright 2011 with permission from Elsevier.

structure (PDB ID 3KBC) was resolved more recently (Reyes et al., 2009), referred to as the
inward-facing conformation, in which the C-terminal core domain moves inwards approximately
18 Å towards the cytoplasm (Fig. 9.14C).
The model which emerges is the following. In the outward-facing carrier, HP2 opens up sponta-
neously to expose the binding site. After glutamate and cotransported ions bind, HP2 closes and
seals the substrate-binding site, and the transporter is in an occluded, outward-facing conformation
(Fig. 9.14D). The whole core domain then moves towards the cytosol which permits the opening of
the internal gate, presumably HP1, and the transporter is in an occluded, inward-facing
 SODIUM/PROTON EXCHANGERS 281

Small intestinal sodium absorption

Brush border
ATP K+
Electrogenic pump

ADP Na+
Na+/glucose
Amino acids

Neutral
NaCl 15 mmol L–1 Na+
– 40 mV

Na+ H+
Cl– HCO3–

FIGURE 9.15
Na absorption in the mammalian small intestine. Neutral Na1 absorption requires Na1/H1 exchangers.

conformation (Fig. 9.14E). Finally, K1 binds to and facilitates reorientation of the carrier back to
an outward-facing state (Fig. 9.14D).

SODIUM/PROTON EXCHANGERS
Intracellular salt and pH must be tightly regulated for cell viability, and therefore organisms, from
all kingdoms of life, require pH-regulated cation/proton antiporters (CPAs) to maintain homoeosta-
sis of H1 and Na1. The carrier-mediated transport of sodium in exchange for protons across mem-
branes is carried out by a family of Na1/H1 exchangers which are often referred to as antiporters.
They are classified as secondary active transporters, since the driving force is the electrochemical
gradient of one of the ions, which drives the counter-transport of the other. They play a varied
number of functions throughout biological systems, and in eukaryotes are vital for cellular homo-
eostasis and play key roles in cancer and heart disease. In humans, there are nine members of the
CPA1 subfamily (designated NHE1-9) and two members of the CPA2 family (NHA1 and NHA2).
The first to be characterized, NHE1, is ubiquitous and localizes to the plasma membrane of most
mammalian cells, and is also found at the basolateral membrane of intestinal enterocytes. In con-
trast, NHE3 is the mammalian epithelial brush border Na/H exchanger. NHE3 is active under basal
conditions and functions as part of neutral NaCl absorption in the intestine and renal proximal
tubule (Fig. 9.15), where it accounts for the majority of total Na absorbed. The best studied Na1/
H1 antiporter is the bacterial NhaA, a member of the CPA2 family, which is an electrogenic anti-
porter with a stoichiometry of 2H1/Na1. The structure of E. coli NhaA has been determined
(Fig. 9.16), and its 384 residues are arranged in 12 transmembrane segments. Human NHA2 is a
poorly characterized Na1/H1 antiporter, which has been recently implicated in essential hyperten-
sion. Based on computer modelling and evolutionary conservation analysis like all of the other
282 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

Cytoplasm

27 Å

Periplasm

FIGURE 9.16
Stereo view of a ribbon representation viewed parallel to the membrane (grey broken lines). The 12 TMSs are
labelled with roman numerals; they comprise the following residues: 12
30 (I), 59
85 (II), 95
116 (III),
121
131 (IVp), 134
143 (IVc), 150
175 (V), 182
200 (VI), 205
218 (VII), 223
236 (VIII), 247
271 (IX),
290
311 (X), 327
336 (XIc), 340
350 (XIp) and 357
382 (XII). N and C indicate the N and C termini.
From Hunte, C., Screpanti, E., Venturi, M., Rimon, A., Padan, E., Michel H., 2005.
Structure of a Na1/H1 antiporter and insights into mechanism of action and regulation by pH. Nature 435, 1197
1202.
Copyright 2005 with permission from Nature Publishing Group.

Na1/H1 exchangers, it is predicted (Fig. 9.17) to have 12 membrane-spanning segments at the N-

terminus (around 500 amino acid residues), with a more hydrophilic C-terminus which is intracellu-
lar and has multiple sites for phosphorylation by protein kinases and binding of other regulatory
factors. Using the crystal structure of the E. coli NhaA (Fig. 9.17), the 3-dimensional structure of
the well-known electroneutral human HNE1 (Landau et al., 2007) and the CPA2 subfamily mem-
ber, human HNA2 (Schushan et al., 2010), has been predicted.

OTHER ROLES OF INTRACELLULAR K1

Quite a number of enzymes are known to be activated by K1
a good example is the glycolytic
enzyme, pyruvate kinase, where the role of the metal is thought to be to orient the phosphoenolpyr-
uvate in the substrate-binding pocket. The more active role of Mg21 in this enzyme is discussed in
Chapter 10, Magnesium
Phosphate Metabolism and Photoreceptors. Since the intracellular concen-
trations of K1 and of Mg21 are high, they dominate metal binding to nucleic acids, with the divalent
Mg21 binding more strongly to the polyanionic sugar-phosphate backbone, on account of its higher
charge. Metal binding reduces electrostatic repulsion between phosphates, stabilizing both base-
pairing and base-stacking; this is underlined by the increase in melting temperature of the DNA in
the presence of metal ions. Much of the intracellular K1 and Mg21 is found bound to ribosomes.
The chromosomes of eukaryotes are linear, and replication of the free ends of these linear DNA
molecules presents particular problems. Human telomeric DNA is typically 5
8 kb long with a
30 single-stranded overhang of 100
200 nucleotides, consisting of tandem repeats of the hexanu-
cleotide sequence d(TTAGGG). Such G-rich sequences can fold into a DNA secondary structure
 Extracellular

(A) NHA2 (B) NhaA (C) NHE1

Cytoplasm

Extracellular

Variable Conserved

FIGURE 9.17
(Upper) The suggested TM topology of NHA2. Residues are coloured according to the hydrophobicity scale of
Kessel and Ben-Tal (Kessel, A., Ben-Tal, N., 2002. Free energy determinants of peptide association with lipid
bilayers. Curr. Top. Membr. 52, 205
253), using the colour bar, with blue through yellow indicating hydrophilic
through hydrophobic. The long loop connecting TM1 and TM2 was omitted for clarity. Overall, the helices are
hydrophobic, but they do feature polar and titratable residues, as anticipated for a transporter. (Lower)
Evolutionary conservation. The evolutionary conservation profiles of NHA2, NhaA, and NHE1, calculated via the
ConSurf Web server (http://consurf.tau.ac.il), (Landau, M., Mayrose, I., Rosenberg, Y., Glaser, F., Martz, E.,
Pupko, T., et al., 2005. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein
structures. Nucleic Acids Res. 33, W299
W302) are shown in (A), (B) and (C), respectively. The intracellular
side is facing up in all panels. The structure of NhaA (Hunte, C., Screpanti, E., Venturi, M., Rimon, A., Padan,
E., Michel H., 2005. Structure of a Na1/H1 antiporter and insights into mechanism of action and regulation by
pH. Nature 435, 1197
1202) and models of NHA2 and NHE1 (Landau, M., Herz, K., Padan, E., Ben-Tal, N.,
2007. Model structure of the Na1/H1 exchanger 1 (NHE1): functional and clinical implications. J. Biol. Chem.
282, 37854
37863) are coloured according to their conservation grades using the colour-coding bar, with
turquoise through maroon indicating variable through conserved. The most variable and most conserved positions
of each transporter are shown as spheres. The three proteins exhibit a highly conserved core, while loops and
lipid-facing residues are variable, as they should.
From Schushan, M., Xiang, M., Bogomiakov, P., Padan, E., Rao, R., Ben-Tal, N., 2010. Model-guided mutagenesis drives functional
studies of human NHA2, implicated in hypertension. J. Mol. Biol. 396, 1181
1196. Copyright 2010 with permission from Elsevier.
284 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

N
H
N
H N
N
N
H
H N N
N N O

H H
O N
M+
N O
H H

N O N N
N H

N H
N
N H
H N

FIGURE 9.18
The structure of a G-quartet.

called G-quadruplexes. Four guanines, interacting via Hoogsteen hydrogen bonds, form a G-quartet
(Fig. 9.18): the cavity in the centre can accommodate a monovalent cation such as Na1 and K1,
with coordination of the four O-6 oxygens of the guanines. The quartets can stack upon each other
to form a multilayer structure, again stabilized by Na1 or K1: this is the G-G-quadruplex.
G-quadruplex nucleic acid structures can form in important regions of the human genome such as
telomeres, gene promoters or transcription start sites, and are involved in several cellular regulation
pathways such as regulation of gene expression and telomere maintenance.
The repetitive G-rich sequences found in the telomeres at the ends of eukaryotic chromosome
can form several isomeric antiparallel arrangements where the tetraplex involves intramolecular
folding, when one polynucleotide supplies two or more strands to the complex. The formation and
stabilization of DNA G-quadruplexes in the human telomeric tandem repeats of the sequence d
(TTAGGG) inhibit the activity of telomerase, a cancer-specific reverse transcriptase which is acti-
vated in 80%
90% of tumours, making it an important target for therapeutic intervention. Clearly,
knowledge of the intact human telomeric G-quadruplex structure under physiological conditions is
a prerequisite for rational, structure-based drug design. NMR spectroscopy has been used to deter-
mine the structures of two human telomeric sequences, Tel22, d[AGGG(TTAGGG)3], and Tel26,
d[AAAGGG(TTAGGG)3AA] in K1 or Na1 solution. Both of these sequences form G-quadruplexes,
as shown for Tel22 in Fig. 9.19. For the extended four-repeat telomeric sequence, Tel26
(Fig. 9.20A), the hybrid-type G-quadruplex structure is the most stable and thus the predominant
form in the presence of K1, regardless of the presence or absence of Na1. Addition of K1 readily
converts the preformed Na1-form G-quadruplex to the hybrid-type G-quadruplex conformation.
Tel26 no longer forms a single stable intramolecular G-quadruplex structure in Na1 solution, likely
 (A) (B)

3⬘
anti anti

syn
3⬘

5⬘
5⬘

FIGURE 9.19
Bis: Folding topologies of Tel22. (A) Propeller-type parallel-stranded intramolecular G-quadruplex in the
presence of K1 in crystalline state (termed hybrid structure). (B) Basket-type mixed parallel/antiparallel-stranded
intramolecular G-quadruplex in Na1 solution determined by NMR.
From Ambrus, A., Chen, D., Dai, J., Bialis, T., Jones, R.A., Yang, D., 2006. Human telomeric sequence forms a hybrid-type
intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution.
Nucleic Acids Res. 34, 2723
2735.

(A) Tel26

Na+ K+

Na+ K+

Other
(B) Tel22 conformations

Na+ K+

Na+ K+

FIGURE 9.20
Schematic diagram of interconversions between the Na1 and K1 forms of telomeric G-quadruplexes.
From Ambrus, A., Chen, D., Dai, J., Bialis, T., Jones, R.A., Yang, D., 2006. Human telomeric sequence forms a hybrid-type
intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution.
Nucleic Acids Res. 34, 2723
2735.
286 CHAPTER 9 SODIUM AND POTASSIUM: CHANNELS AND PUMPS

(A) (B) (C) (D) (E) (F)

FIGURE 9.21
Structures and their respective schematic topologies of (A) the hybrid-1 24TTG, (B) the hybrid-2 26TTA, (C) the
antiparallel 2-quartet 22GT, (D) the antiparallel in sodium 22AG, (E) the antiparallel 2-quartet 22CTA, and (F)
the parallel Pu24. These structures were obtained by NMR in 100 mM K1 except for 22AG whose structure was
obtained in 100 mM Na1.
From Marchand, A., Gabelica, V., 2016. Folding and misfolding pathways of G-quadruplex DNA. Nucleic Acids Res. 44,
10999
11012. Copyright 2016 with permission from OUP.

caused by the steric interference of the flanking sequences with the diagonal loop, both of which are
positioned on the same side of the basket-type G-quadruplex structure. The truncated Tel22
(Fig. 9.20B) forms a single stable basket-type intramolecular G-quadruplex in Na1 solution.
However, in the presence of K1, Tel22 does not form a single G-quadruplex structure and is likely to
have two stable G-quadruplex conformations coexisting. Addition of K1 to the preformed Na1
basket-type G-quadruplex readily converts the conformation to the K1-form, thus the two intercon-
vertible K1 G-quadruplex conformations are both more stable than the Na1-basket-type
G-quadruplex. A more recent study of the human telomeric sequence (Marchand and Gabelica,
2016), with the repetitive 30 -end sequence d(TTAGGG)n, has shown that G-quadruplexes fold via
branched pathways with multiple parallel reactions, as illustrated by the structures presented in
Fig. 9.21. Clearly, establishing the distinct folding topology of the telomeric G-quadruplex which is
found under physiological conditions could make it an attractive target for specific targeting by small
molecular weight drugs which, by stabilizing the telomeric G-quadruplexes, could represent an
important cancer therapeutic strategy.

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