Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

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charged amino acids (aspartates) denominated the

“Ca

2þ

bowl.” The Ca

2þ

bowl has emerged as a good

candidate for the high-afﬁnity Ca

2þ

-binding site.

Slo channels control a large variety of physiological

processes, including smooth muscle tone, neurosecretion,

and hearing. Despite being coded by a single gene, the

diversity of Slo channels is great. Regulatory

-subunits,

splicing, and metabolic regulation create this diversity

fundamental to the adequate function of many tissues.

Except in heart myocytes, Slo channels are almost

ubiquitously expressed among mammalian tissues, and

they play a variety of roles. In smooth muscle, local Ca

2þ

increases, called Ca

2þ

sparks, generate spontaneous

transient outward currents, which are generated by Slo

channels. This hyperpolarizes the membrane causing

muscle relaxation. In chromafﬁn cells, Slo channels are

important for rapid termination of the action potential.

In the cochlea of turtles, frogs and chicks, gradients of

spliced Slo channels, and

-subunits through an

interplay with voltage-dependent Ca

2þ

channels, they

are able to generate the different oscillatory responses in

the hair cells.

KCNQ CHANNELS

The ﬁrst member of the KCNQ family to be cloned,

KCNQ1, was isolated by virtue of the fact that

mutations in this gene give rise to the most common

form of long QT syndrome, LQT1. Exploiting their

homology to KCNQ1, other members of this family

were subsequently cloned. KCNQ1 encodes the major

subunit of the cardiac I

channel, which is involved in

repolarization of the ventricular action potential.

KCNQ1 mRNA is expressed strongly in the heart,

with lower levels in pancreas, kidney, lung, placenta,

and ear.

It has been suggested that KCNQ2 and KCNQ3 is a

heteromer, an idea which is consistent with their

overlapping tissue distribution and the fact that

antibodies directed against KCNQ2 are able to

coimmunoprecipitate KCNQ3, and vice versa. The

heteromeric KCNQ2/KCNQ3 channel corresponds to

neurone’s M-channel. They are widely distributed

throughout the brain, being expressed at high levels

in hippocampus, chordate nucleus, and amygdala.

Mutations in KCNQ2 and KCNQ3 rise to a form of

idiopathic epilepsy known as benign familial neo-

natal convulsions.

CHANNELS

The vertebrate fast voltage-gated K

delayed rectiﬁer

channels fall into a variety of families (K

1–K

11)

according to their amino acid sequence similarity

(Figure 2). A channel from a fruit ﬂy Drosophila was

the ﬁrst K

channel to be cloned (denominated in

vertebrates as K

1.1, the ﬁrst number denoting

subfamily and the second, order of discovery). The

identiﬁcation started with mutant ﬂies called shakers,

which shake their legs while under ether anesthesia.

These ﬂies lack a fast transient K

current in

presynaptic terminals, so repolarization of the action

potential is delayed and the evoked release of

neurotransmitter from a single action potential

becomes enormous.

channels are found in a wide range of different

tissues and they have many different functions. Some

ideas of the diversity of roles of K

channels are shown in

Figure 2. For example, K

3.1 channels are found in some

neurons that are specialized to ﬁre very short action

potentials at high rates, such as those of the auditory

system; K

1.3 channels are found in leukocytes playing a

role in inﬂammatory response; K

1.2 channels are found

in smooth muscle participating in contractil tone

regulation; and K

4.2 channels are expressed in myo-

cardium and their function is action potential pro-

longation in heart.

Defects in K

channels function therefore may

have profound physiological effects. Linkage studies

have identiﬁed the gene responsible for episodic ataxia

type-1. This gene encoded for K

1.1 channel, which is

expressed in the synaptic terminals and dendrites in

brain neurones.

FURTHER READING

Hille, B. (2001). Ions Channels of Excitable Membranes. Sinauer

Associates, Sunderland, Massachusetts.

Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chalt, B. T., and

MacKinnon, R. (2003). X-ray structure of a voltage-dependent K

channel. Nature 423, 33– 41.

Laine

,M.,Papazian,D.M.,andRoux,B.(2004).Critical

assessment of a proposed model of Shaker. FEBS Lett. 564,

257–263.

VOLTAGE-DEPENDENT K

CHANNELS 403

Orio, P., Rojas, P., Ferreira, G., and Latorre, R. (2002). New disguises

for and old channel: MaxiK channel

-subunits. News Physiol. Sci.

17, 156–161.

BIOGRAPHY

Ramon Latorre is Professor and Chair of the Laboratory of Biophysics

and Molecular Physiology, CECS. He holds a Ph.D. from the

University of Chile and received his postdoctoral training at the

Biophysics Laboratory in the National Institutes of Health, Maryland.

He is a Foreign Associate of the National Academy of the United States

of America. The main scientiﬁc interest of Dr. Latorre is the molecular

workings of ion channels.

Francisco Morera holds a degree in biochemistry from the Catholic

University, Chile and is at present a Ph.D. in the Austral University,

Valdivia, Chile.

404 VOLTAGE-DEPENDENT K

CHANNELS

Voltage-Sensitive Ca

2þ

Channels

Harald Reuter

University of Bern, Bern, Switzerland

Ion channels are integral, pore-forming transmembrane

proteins that serve to pass ions across biological membranes.

The channels differ in their ion selectivity, i.e., each type allows

certain ion species to permeate through the open channel pore.

Conformational changes of the proteins leading to openings or

closings of the channels are called “gating.” It is achieved

either by binding of ligands to special sites on the channels

(ligand-gated ion channels), or by changes in membrane

potential (voltage-gated ion channels). Calcium (Ca

) chan-

nels are voltage-gated and have a high selectivity for Ca

ions.

Opening of these channels by membrane depolarization leads

to an inﬂux of Ca

ions into the cell. The resulting increase in

the intracellular Ca

concentration initiates essential physio-

logical functions, such as cardiac contraction, secretion of

hormones and neurotransmitters, gene transcription, or

repetitive electrical activity of neurons and the heart. Most of

these functions result from binding of Ca

to speciﬁc

intracellular proteins (e.g., calmodulin, troponin C) which

subsequently mediate the Ca

-dependent cellular responses.

The diversity of voltage-gated Ca

channels in surface

membranes of different cells is important for initiation of

speciﬁc signaling cascades within the cells. The various types of

channels are encoded by different genes and show tissue-

speciﬁc expression. They are targets for toxins and for drugs

used for therapeutic purposes.

Molecular Properties

STRUCTURES

Voltage-gated Ca

2þ

channels are oligomeric protein

complexes composed of the voltage-sensitive, pore-

forming

-subunits, together with auxiliary disulﬁde-

linked

-subunits and intracellular

-subunits

(Figure 1). An additional transmembrane

-subunit is

associated with

S in skeletal muscles. The diversity of

2þ

channel types not only results from different genes

coding for the different channel subunits (10 for

, 4 for

d, and 4 for

), but also from multiple splice variants

of the subunits. The

-subunits consist of four

homologous domains (I–IV), each being composed of

six transmembrane

-helical segments (S1–S6). The

N and C termini are located intracellularly, and

the domains are linked together by intracellular loops.

In the folded structures of

-subunits, hairpin loops

between transmembrane segments S5 and S6 constitute

the channel pore. Segments S4 contain highly conserved,

positively charged amino acids that act as voltage-

sensitive sensors during gating of the channels. The

-subunits are involved in the regulation of the

channels’ gating properties and, like

d-subunits, play

a role in the functional expression of the channels in cell

surface membranes.

SELECTIVITY

2þ

channels are selective pathways for the movement

of Ca

2þ

ions down their electrochemical gradient into

the cell. The pore-forming structures of the channel

contain four negatively charged glutamate residues, each

being in a homologous position on the four domains.

The resulting ring of negative charges facing the pore

coordinates binding of an entering Ca

2þ

ion. A second

2þ

ion that enters the channel reduces the binding

afﬁnity of the ﬁrst one by electrostatic repulsion, thus

kicking it off from its binding site and letting it

move down its electrochemical gradient. Thus, the

selectivity of the channel for Ca

2þ

is critically dependent

on the correctly placed negative charges of glutamate

in the pore.

CALMODULIN BINDING

Two forms of autoregulation, Ca

2þ

-dependent inacti-

vation and facilitation of Ca

1.2 channels (Table I),

involve calmodulin (CaM). The subunit

1C of Ca

1.2

contains a consensus CaM-binding isoleucine-glutamine

(IQ)-motif in its cytoplasmic C-terminal tail. The IQ-

motif consists of 12 amino acids. Extensive mutations

within this sequence revealed structural determinants

for the two opposing forms of channel regulation. Two

short stretches of aminoacids upstream of the IQ-motif

seem to be sites for constitutive, Ca

2þ

-independent

binding of apoCaM. The IQ-motif binds CaM only in

the presence of Ca

2þ

. It represents the regulatory site for

2þ

-dependent inactivation, while facilitation may

require additional activation of Ca/CaMkinaseII.

PHOSPHORYLATION

2þ

channel function can be modulated by second

messenger-activated protein kinases (PKs) such as

cyclicAMP-dependent PKA, diacylglycerol-dependent

PKC, or Ca/calmodulin-dependent kinases. Although

the exact molecular mechanisms of these modulatory

pathways are not yet fully understood, they do depend

on phosphorylation by kinases of speciﬁc aminoacid

residues of the Ca

2þ

channel protein. Multiple intra-

cellular sites of phosphorylation have been identiﬁed on

- and

-subunits. However, which phosphorylation

sites are necessary and sufﬁcient for different forms of

modulation of Ca

2þ

channel function by the various

protein kinases has not been clearly resolved.

GPROTEINS

2þ

channels of the Ca

2 family (Table I) that are

involved in neurotransmitter and hormone secretion are

inhibited by G proteins. There is excellent evidence that

the rapid inhibition of the channels results from direct

binding of the

-subunits of G proteins to an identiﬁed

segment of the intracellular loop between domains I and

II (Figure 1), but other sequences in the C and N termini

of the

-subunits may also be important. Rapid

inhibition of the channels by this mechanism does not

require formation of soluble intracellular messengers.

The inhibition results from a membrane-delimited

pathway leading to the release of the

-subunit of the

G-protein complex.

-Currents

PHYSIOLOGY

Depolarization of cell membranes during excitation

leads to spontaneous openings (activation) and closings

(inactivation) of Ca

2þ

channels. At a given voltage,

stochastic openings of multiple channels produce a

considerable ﬂow of Ca

2þ

ions into a cell and can thus be

measured as an electrical current. The currents are

recorded by electrophysiological methods, at a level of

resolution where even the current through an individual

channel can be measured (patch-clamp method). This

allows detailed quantitative analyses of the kinetic

properties (gating) and selectivities of the channels.

Currents through different types of Ca

2þ

channels

have traditionally been classiﬁed as L-, P/Q-, N-, R-,

and T-type (Table I). This classiﬁcation resulted from

functional and pharmacological analyses before the

channels were cloned and sequenced. A general over-

view of the distribution of the channels in different

tissues is summarized in Table I. The speciﬁc expression

of different Ca

2þ

channel types in different tissues is of

FIGURE 1 Oligomeric subunit complex of voltage-gated Ca

2þ

channels. The subunit composition consists of the pore-forming

1-subunit with

its four homologous domains, the intracellular

-subunit, and the disulﬁde-linked a2-subunit. The sites of interaction between the subunits are

indicated by the squares. An extra

-subunit is present in skeletal muscle Ca

1.1 complex (inset). Kindly prepared by Dr. R. D. Zuehlke, Bern.

406 VOLTAGE-SENSITIVE CA

CHANNELS

great physiological and pharmacological importance.

The localization of individual channels in association

with other cellular constituents governs speciﬁc signal-

ing pathways. For example, P/Q-,and N-type channels

are directly linked to the SNARE (Soluble N-ethylma-

leimide sensitive fusion protein Attachment protein

REceptor) protein complex in neural and endocrine

cells that is essential for secretion of neurotransmitters

and hormones. L-type channels that are predominant in

cardiac cells, are in close proximity to intracellular Ca

2þ

stores. When Ca

2þ

ions move through the channels

into these cells during excitation, they release more Ca

2þ

from the stores which eventually leads to contraction of

the heart. Thus, changes in the intracellular Ca

2þ

concentration are coding for important information of

diverse cellular functions. Autoregulatory mechanisms,

by which the channels inactivate in voltage- and Ca

2þ

dependent manners, prevent Ca

2þ

overload of the

cells. Ca

2þ

-dependent inactivation has been studied

most extensively in L-type Ca

2þ

channels, but

may also apply to other channel types. During the

ﬂow of Ca

2þ

ions through the pore, they bind to

constitutively tethered apoCaM at the cytoplasmic C

terminus of the channel. Occupancy of CaM by Ca

2þ

triggers binding of its C-terminal lobe to the

nearby IQ-motif and accelerates inactivation of the

channel, possibly by disinhibiting voltage-dependent

inactivation (Figure 2).

FIGURE 2 Model of voltage- and Ca

2þ

-dependent gating of a Ca

2þ

channel. The model shows a schematic folding of the

1-subunit in Figure 1.

At membrane resting potential the pore is closed (resting state); upon depolarization (DV) the pore opens and Ca

2þ

ions move across the membrane

down their electrochemical gradient (open state); when the intracellular Ca

2þ

-concentration increases, Ca

2þ

binds to tethered calmodulin

(CaM) which then attaches itself to the regulatory IQ-motif and promotes closure of the pore (inactivated state). Kindly prepared by Dr. R. D.

Zuehlke, Bern.

TABLE I

Channels

Channel

types

Pore-

forming

-subunits

Tissue

distribution

Pharmacology

(blockers)

HVA

1.1 (L)

1S Skeletal muscle DHPs

1.2

1C Heart, smooth

muscle, CNS

1.3

1D CNS, kidney,

cochlea, glands

1.4

1F Retina

2.1 (P/Q)

1A CNS

-Agatoxin

2.2 (N)

1B CNS

-Conotox.GVIA

2.3 (R)

1E CNS, heart,

glands

LVA

3.1 (T)

1G CNS, sinus node Mibefradil

3.2

1H CNS, heart,

kidney, liver

3.3

1I CNS

Channel types: HVA ¼ high-voltage-activated channels; LVA ¼

low-voltage-activated channels; letters in brackets refer to the

nomenclature of currents carried through the different channel types.

Pore-forming

-subunits encoded by identiﬁed genes have

multiple splice variants (not shown); CNS ¼ central nervous system;

DHPs ¼ Dihydropyridines.

VOLTAGE-SENSITIVE CA

CHANNELS 407

MODULATION

Modulation of Ca

2þ

channel activity by neurotransmit-

ters and hormones has become a major ﬁeld of research.

The up-regulation of cardiac Ca

2þ

current (Ca

1.2) by

epinephrine was the ﬁrst example of modulation of an

ion channel by a neurotransmitter. It has been shown to

be the result of activation of the cyclicAMP pathway

via

-adrenoceptors and subsequent phosphorylation

by protein kinase A of aminoacid (serine) residues

at intracellular sites of the channel. This causes

enhanced probability of openings of the channels,

resulting in largely increased Ca

2þ

currents. Other

examples of modulation mainly concern N- and

P/Q-type Ca

2þ

channels (Ca

2.1 and Ca

2.2; Table I).

They are inhibited by neurotransmitters, such as

norepinephrine. Release of G protein

-subunits by

neurotransmitters shifts channel activation into a

reluctant state, thus causing a reduction of Ca

2þ

currents

during depolarization. These are just two examples of

modulation of Ca

2þ

channel activities. There are

several others that are not included under the scope of

this article.

PHARMACOLOGY

2þ

channels are important targets for toxins and

drugs. Although a full discussion of their effects is

beyond the scope of this article, a few of them are

listed in Table I. Dihydropyridines (e.g., nifedipine,

nimodipine) are relatively speciﬁc inhibitors of the

1 channel family, mainly Ca

1.2 type. Other drugs

acting on these channels are the phenylalkyamines

verapamil and the benzothiazepine diltiazem. They are

grouped together as “Ca-channel blockers,” and are

therapeutically used for the treatment of cardiovas-

cular diseases, notably hypertension and angina

pectoris. The toxins have no, or very limited,

therapeutic use. In some cases of otherwise untrea-

table pain, the N-type channel blocker

-conotoxin

GVIA has been successfully applied. Because of

unwanted side effects, the T-type channel blocker

mibefradil has been withdrawn from therapeutic use,

although it was quite effective in the treatment of

essential hypertension.

FURTHER READING

Catterall, W. A. (2000). Structure and regulation of voltage-gated Ca

2þ

channels. Annu. Rev. Cell Dev. Biol. 16, 521– 555.

Hille, B. (2001). Ionic Channels of Excitable Membranes. Sinauer,

Sunderland, MA.

McDonald, T. F., Pelzer, S., Trautwein, W., and Pelzer, D. J. (1994).

Regulation and modulation of calcium channels in cardiac,

skeletal, and smooth muscle cells. Physiol. Rev. 74, 365–507.

Perez-Reyes, E. (2003). Molecular physiology of low-voltage-activated

T-type calcium channels. Physiol. Rev. 83, 117–161.

Reuter, H. (1996). Diversity and function of presynaptic calcium

channels in the brain. Curr. Opin. Neurobiol. 6, 331– 337.

Saimi, Y., and Kung, C. (2002). Calmodulin as an ion channel subunit.

Annu. Rev. Physiol. 64, 289–311.

BIOGRAPHY

Harald Reuter is Professor Emeritus and former Director of the

Institute of Pharmacology at the University of Bern, Switzerland. He

received his M.D. at the University of Mainz, Germany. Dr. Reuter ﬁrst

described Ca

2þ

currents and the sodium/calcium-exchange transport in

the heart, and discovered regulation of Ca

2þ

channels by catechol-

amines and calmodulin. He is further interested in synaptic function

in the brain. He has received many awards for his discoveries and

has been elected a Foreign Associate of the National Academy of

Sciences, USA.

408 VOLTAGE-SENSITIVE CA

CHANNELS

Voltage-Sensitive Na

Channels

William J. Brammar

University of Leicester, Leicester, UK

Voltage-sensitive sodium channels are the transmembrane

protein complexes that mediate the increased permeability to

ions during the initial rising phase of the action potential

in most excitable cells. The channels are closed at resting

membrane potentials, but open in response to membrane

depolarization to become selectively permeable to Na

ions.

The open or activated state is transitory: channels are

inactivated and Na

permeability returns to the baseline

level within about a millisecond, allowing the repolarization

phase of the action potential that is controlled by K

channels.

A family of genes encoding multiple sodium channel

isoforms has been revealed by molecular cloning. Mutations in

genes encoding Na

channel subunits give rise to genetic

diseases affecting the function of skeletal muscles, the heart or

the brain. Sodium channel proteins include target-sites for the

action of a number of neurotoxins, insecticides, and local

anesthetics.

Isolation and Puriﬁcation

of Sodium Channel Proteins

The ﬁrst biochemical identiﬁcation of sodium channel

proteins relied on speciﬁc covalent labeling of the

proteins with a photoreactive derivative of the north

African scorpion toxin. Denaturing polyacrylamide gel

electrophoresis of proteins derived from rat brain

synaptosomes revealed two labeled polypeptides of

260 and 36 kDa that were subsequently designated as

the

-and

1-subunits of the sodium channel. Puriﬁ-

cation of the sodium channel solubilized from rat

brain revealed three polypeptides,

of 260 kDa,

1of

36 kDa and

2 of 33 kDa, in a 1:1:1 stoichiometry.

The

1-subunit is non-covalently associated with the

-subunit, whereas the

2-subunit is covalently attached

via disulﬁde bonds.

The sodium channels puriﬁed from rat skeletal

muscle sarcolemma or transverse tubules consist of a

260 kDa subunit and one or two subunits of 38 kDa.

In contrast, those puriﬁed from eel electroplax and

chicken heart contain only the 260 kDa component.

Reconstitution experiments, in which the puriﬁed

proteins were incorporated into phospholipid vesicles

to generate fully functional, neurotoxin-sensitive

sodium channels, proved the validity of the protein

puriﬁcation methods based on neurotoxin-binding.

Primary Structures of Sodium

Channel Proteins

THE

-SUBUNITS OF SODIUM CHANNELS

Sequences encoding a complete Na

channel protein

were ﬁrst cloned as cDNAs derived from the mRNA of

the electroplax of the electric eel. The deduced sequence

of 1820 amino acids contains four internally homolo-

gous domains, each having six putative membrane-

spanning

-helical segments (Figure 1). In each

homology unit, ﬁve segments (S1, S2, S3, S5, and S6)

are predicted to be hydrophobic, membrane-spanning

helices. The interspersed S4 transmembrane segments

contain positively charged amino acids located at every

third position, forming an

-helix with a spiral of

positive charges.

The suggestion that the four S4 segments act as

voltage-sensors for the gating of the Na

channel has

been conﬁrmed by mutational alteration of the posi-

tively charged residues. The absence of an N-terminal

signal sequence suggests that the N terminus is

cytoplasmic.

The structural motif of four internally homologous

domains, ﬁrst promulgated for the sodium channel of

the eel electroplax, is also evident in the voltage-gated

calcium and potassium channels and in other channels

within the large superfamily. Discernable sequence

similarity (, 32–37% identity) between the four hom-

ologous domains of the voltage-gated Na

channels and

those of the voltage-gated Ca

2þ

channels reﬂects the

common ancestry of these two channel types.

Sequences encoding mammalian sodium channel

-subunits have been isolated using segments of the

eel electroplax Na

channel cDNA as hybridization

probes and by screening expression libraries with

antibodies prepared against puriﬁed components.

DNA sequences encoding regions of brain Na

channels

have been used in low stringency hybridizations to

isolate cDNAs encoding

-subunits from other tissues

such as skeletal muscle and heart. Although the different

channel

-subunits vary slightly in length and sequence,

they are clearly homologous and share fundamentally

similar structures.

THE

-SUBUNITS OF SODIUM CHANNELS

Both the

1 and

2 subunits of the voltage-sensitive Na

channel are predicted to contain a single transmembrane

region, located near the intracellular C terminus,

and an extracellular domain containing a single

“immunoglobulin-like fold” (Figure 1). The latter

consists of two superimposed

-sheets held together by

hydrophobic interactions and is characteristic of the

family of cell adhesion molecules that mediate cell–cell

interactions. In such molecules, immunoglobin-like

folds bind extracellular proteins and inﬂuence inter-

actions with the extracellular matrix and with other cells

to affect cell shape and mobility. Rat brain Na

channels

and the

2-subunit have been shown to bind to the

extracellular matrix proteins tenascin-C and tenascin-R,

leading to the suggestion that such interactions may

inﬂuence the formation of specialized areas of high Na

channel density such as nodes of Ranvier.

Heterologous expression of cDNAs encoding Na

channel

and

subunits has shown that the

1 and

subunits modulate channel-gating behavior. Expression

of the unaccompanied

-subunit of brain or skeletal

muscle Na

channel in Xenopus oocytes produces

channels that activate and inactivate more slowly

than the native channels. Coexpression of

1 and

subunits with the

-subunit yields channels that gate

normally, and the accelerating effect of the

1-subunit is

mediated by the immunoglobulin-like fold in the extra-

cellular domain.

The Molecular Basis of Sodium

Channel Function

THE OUTER PORE AND

SELECTIVITY FILTER

The guanidinium-containing compounds tetrodotoxin

and saxitoxin, selective and reversible blockers of Na

channels at nanomolar concentrations, have been

inﬂuential in allowing identiﬁcation of the outer pore

and selectivity ﬁlter of the channel. These toxins are

membrane-impermeant and act only from the extra-

cellular side of the membrane to plug the selectivity ﬁlter

in the outer pore of the channel. Mutational studies have

identiﬁed a pair of amino acids, mostly negatively

charged, in analogous positions in all four domains of

FIGURE 1 Primary structures of the subunits of the voltage-gated sodium channel. The

1, and

2 polypeptide chains are shown to illustrate

their relationship to each other and to the cell membrane. The cylinders represent putative

-helices. The lengths of the intracellular and

extracellular loops are roughly proportional to the number of amino acid residues in the brain sodium channel subtypes. The extracellular domains

of the two

subunits are drawn as immunoglobulin-like folds. C shows the site of probable N-linked glycosylation: P in red circles, sites of

phosphorylation by (circles) and PKC (diamonds); the green regions are pore-lining segments; white circles represent the outer and inner rings of

amino acid residues that form the ion selectivity ﬁlter and the tetrodotoxin-binding site: yellow shows the S4 voltage sensors; h in a blue circle

represents the inactivation particle and blue circles the sites forming the receptor for the inactivation gate. Binding sites for

and

scorpion toxins

(

-ScTx and

-ScTx) are shown by the downward arrows. (Reproduced from Catterall, W. A. (2000). From ionic currents to molecular mechanism:

The structure and function of voltage-gated sodium channels. Neuron 26, 13–25, with permission from Elsevier).

410 VOLTAGE-SENSITIVE NA

CHANNELS

the rat brain Na

channel

-subunit, that are important

for binding tetrodotoxin and saxitoxin (Figure 1). These

four pairs of amino acids were postulated to form outer

and inner rings that acted as both the receptor-site for

tetrodotoxin and saxitoxin and the selectivity ﬁlter in

the outer pore of the channel. The concept is strongly

supported by the ﬁnding that replacement of the four

amino acid residues in the inner ring by four glutamic

acid residues, their counterparts in voltage-gated Ca

2þ

channels, made the Na

channels calcium-selective.

Cardiac Na

channels bind tetrodotoxin with an

afﬁnity that is 200-fold lower than that of the brain or

skeletal muscle channels, because of a replacement of an

aromatic amino acid by a cysteine residue in the outer

pore region of domain I. Cadmium is a high-afﬁnity

blocker of cardiac Na

channels because of its inter-

action with this same cysteine residue. Electrophysio-

logical measurements of the voltage-dependence of the

cadmium block of cardiac Na

channels place the target

cysteine residue, and thus the selectivity ﬁlter, about 20%

of the way through the membrane electric ﬁeld.

THE INNER PORE

Local anesthetics, such as lidocaine and procaine, are

generally lipid-soluble tertiary amine compounds that

inhibit propagated action potentials by blocking Na

channels. A quaternary amine analogue of lidocaine,

QX-314, which is positively charged at all pHs and not

lipid-soluble, blocks Na

channels only when applied to

the inside of the membrane. Blockage requires prior

opening of the channels by depolarization. Mutagenesis

studies demonstrated that the binding-site for local

anesthetics lies in the IVS6 transmembrane segment

(Phe1764 and Tyr1771 in rat brain type IIA channels).

Similar amino acid residues in the S6 transmembrane

segment of certain voltage-gated K

channels act as

the targets for tetraethylammonium-based blockers.

These amino acids are located in an aqueous cavity

within the inner pore region of the K

channel, by

extrapolation from a bacterial K

channel whose

quaternary structure has been determined.

THE MECHANISM OF

VOLTAGE-DEPENDENT ACTIVATION

The voltage-dependent activation of sodium channels

involves the net outward movement across the mem-

brane electric ﬁeld of , 12 electronic charges in the

channel protein. The S4 segments of the

-subunits, with

positively charged amino acids at every third position,

were predicted to act as the voltage-sensor and to

undergo outward movement on depolarization of the

membrane, initiating a conformational change associ-

ated with pore-opening.

Mutagenesis studies showed that neutralization of the

positive charges in the S4 domains reduced the steepness

of the potential-dependence of activation, equivalent to

reducing the gating charge. Physical movement of the S4

segments has been demonstrated by substituting

cysteine for the basic amino acids, then assessing the

availability of the individual cysteine-SH groups to

chemical modiﬁcation by membrane-impermeant sulf-

hydryl reagents, before and after depolarization. Three

successive basic amino acids in the S4 segment of

domain IV (Arg1448, Arg1451, and R1454) move from

being inaccessible within the membrane to being

available for reaction from outside the membrane.

Similar outward movements of positively charged S4

segments have been demonstrated for voltage-sensitive

channels. In this case it has been shown by

mutagenesis that the positive charges in S4 are paired

with negatively charged residues in either S2 or S3

segments, stabilizing the S4 segments in the membrane.

THE BASIS OF INACTIVATION

Native sodium channels spontaneously inactivate within

milliseconds of opening. The sensitivity of this rapid

inactivation to limited cytoplasmic applications of

proteases lead to the proposal of the ‘ball and chain’

hypothesis of inactivation, in which an intracellular

inactivation gate (‘the ball’), tethered by a ﬂexible

‘chain’, was able to interact with the intracellular

mouth of the pore and block the channel. The

inactivation gate has been localized to a short intra-

cellular loop connecting regions III and IV. Antipeptide

antibodies directed against this sequence, cutting the

loop in this region and mutagenesis of a hydrophobic

triad of Ile, Phe, and Met in the region all prevent fast

inactivation. Voltage-dependent movement of this seg-

ment has been detected by the depolarization-induced

loss of accessibility to cytoplasmically applied reagents.

The receptor for the inactivation gate has been

delineated by scanning mutagenesis experiments. Mul-

tiple amino acid residues at the intracellular end of

transmembrane segment IVS6 and in intracellular loops

IIIS4-S5 and IVS4-S5 contribute to the receptor for the

inactivation the gate, allowing its binding to block the

pore of the channel (Figure 1).

Modulation of Channel Activity

Sodium channels in neurons and skeletal muscle are

susceptible to modulation by several protein kinases.

Cyclic AMP-dependent protein kinase A (PKA) phos-

phorylates sodium channel

-subunits in brain synapto-

somes and intact neurons on four sites in the

intracellular loop between domains I and II. The

consequence of phosphorylation by PKA is a reduction

VOLTAGE-SENSITIVE NA

CHANNELS 411

in sodium conductance, with little effect on the voltage-

dependence of activation and inactivation. Activation of

adenylate cyclase via dopamine acting at D1-like

receptors reduces action potential generation and peak

sodium currents in hippocampal neurons. The presence

of a protein kinase-anchoring protein, AKAP-15,

which brings PKA close to the sodium channel, is

essential for this regulation. Cocaine, which increases

dopaminergic neurotransmission by inhibiting dopa-

mine-uptake, also reduces sodium currents in nucleus

accumbens neurons.

Protein kinase C (PKC) also acts on sodium channel

-subunits, slowing channel inactivation and reducing

peak sodium currents. Phosphorylation of a site in the

inactivation gate is responsible for the slowing of channel

inactivation. The reduction in peak current is a con-

sequence of phosphorylation at sites in the intracellular

loop between domains I and II (see Figure 1).

The effects of PKA are enhanced by PKC-dependent

phosphorylation and by steady membrane depolariz-

ation, so that the information from three distinct

signaling pathways are integrated via the sodium

channels in the hippocampus and CNS.

The Genetics of Sodium Channels

THE GENE– PROTEIN FAMILY

SODIUM CHANNELS

A large family of sodium channel genes and proteins,

containing at least ten members, has been recognized

through molecular cloning and heterologous expres-

sion to make characterizable protein products. The

nomenclature for sodium channels has been informal

and inconsistent, but a standardized system has recently

been proposed by a consortium of leading researchers

(see Table I).

The nine isoforms of the sodium channel

-subunit

that have been characterized are greater than 50%

identical in the transmembrane and extracellular

domains. The continuously variable pattern of

sequences is consistent with their constituting a single

family. The Na

1.1, Na

1.2, Na

1.3, and Na

1.7

-subunits are the most closely related. These four

channels are all highly tetrodotoxin-sensitive and

expressed in the central and peripheral nervous system.

The corresponding genes are all located on human

chromosome 2q23–24. The Na

1.5, Na

1.8, and

1.9 channel

-subunits, all forming tetrodotoxin-

resistant sodium channels, are produced in heart and

neurons of the dorsal root ganglia from genes located on

chromosome 3p21–24. The skeletal muscle sodium

channel isoform, Na

1.4, formerly called type

1, is at

least 84% identical to the group of channels encoded on

chromosome 2, but has a more distant phylogenetic

relationship as determined by parsimony comparison.

Comparison across mammalian species shows that

the chromosome segments containing sodium channel

-subunit genes are paralogous segments containing

many sets of related genes, generated by large-scale

duplication events during early vertebrate evolution.

Several sodium channel

-subunits have been recog-

nized by molecular cloning and DNA sequencing, but

have not yet been characterized by functional expression

(Table I,Na

). These sequences have more than 80%

identity to each other and are phylogenetically closely

related to the group of sodium channel subunits encoded

on human chromosome 2q23 –24, where the human

SCN6A gene is located.

The genes encoding mammalian sodium channel

-subunits are complex, with more than 20 exons.

Splice variants of several sodium channel proteins have

been identiﬁed and more are likely to be found.

GENETIC DEFECTS AFFECTING

VOLTAGE-GATED SODIUM CHANNELS

Mutations in genes encoding sodium channel subunits

are associated with several inherited human diseases

characterized by hypersensitivity, including several

monogenic epilepsy syndromes. Mutations in the

human SCN1A gene have been associated with

familial generalized epilepsy with febrile seizures plus

(GEFSþ), an autosomal dominant syndrome character-

ized by febrile seizures (FS) and a variety of afebrile

generalized seizure types. De novo mutations in SCN1A

are a major cause of severe myoclonic epilepsy of

infancy (SMEI or Dravet syndrome). Benign familial

neonatal-infantile seizures have been ascribed to

mutations in the SCN2A gene.

Mutations in the SCN4A gene encoding the skeletal

muscle sodium channel

-subunit are the cause of the

hereditary disorders of sarcolemmal excitability, hyper-

kalemic periodic paralysis (HYPP or hyperPP) and

paramyotonia congenita (PMC). Mutations have been

shown to cause reduced rates of channel inactivation,

altered voltage-sensitivity or slowed coupling between

activation and inactivation. HYPP is a muscle disorder

that is prevalent in American quarter horses, where

selective breeding for muscular deﬁnition and hypertro-

phy has favored spread of the mutant SCN4A gene in the

gene-pool.

One of the causes of long QT syndrome, an inherited

cardiac arrhythmia, is mutation in the SCN5A gene,

leading to non-inactivating sodium currents that

prolong the plateau of the cardiac action potential.

The result is an elongated interval between the QRS

complex and the T wave in the electrocardiogram. The

mutations usually affect amino acid residues in the

inactivation gate or its receptor region, reducing the rate

of inactivation.

412

VOLTAGE-SENSITIVE NA

CHANNELS