Review article

Channelopathies: ion channel defects linked toheritable clinical disorders

Ricardo Felix

AbstractElectrical signals are critical for the func-tion of neurones,muscle cells, and cardiacmyocytes. Proteins that regulate electricalsignalling in these cells, including voltagegated ion channels, are logical sites whereabnormality might lead to disease. Ge-netic and biophysical approaches arebeing used to show that several disordersresult from mutations in voltage gated ionchannels. Understanding gained fromearly studies on the pathogenesis of agroup of muscle diseases that are similarin their episodic nature (periodic paraly-sis) showed that these disorders resultfrom mutations in a gene encoding a volt-age gated Na+ channel. Their characteri-sation as channelopathies has served as aparadigm for other episodic disorders.For example, migraine headache andsome forms of epilepsy have been shownto result from mutations in voltage gatedCa2+ channel genes, while long QT syn-drome is known to result from mutationsin either K+ or Na+ channel genes. Thisarticle reviews progress made in the com-plementary fields of molecular geneticsand cellular electrophysiology which hasled to a better understanding of voltagegated ion channelopathies in humans andmice.(J Med Genet 2000;37:729740)

Keywords: ion channel genetics; ion channel physiopa-thology; channelopathies; hereditary diseases

Many interesting advances in molecular medi-cine over the last few years have come fromresearch in molecular genetics. Virtually everymonth novel genes linked to diVerent clinicaldisorders are cloned. Sometimes these findingsrelate to common diseases, while other timesthey concern diseases that are fairly rare. In anycase, the information often provides importantinsight into mechanisms underlying a particu-lar disease, or new means of understanding thefunction of a particular protein. A good exam-ple of this is the field of ion channel research. Inparallel with the progress in the understandingof the structure and function of these proteins,the list of genetic diseases linked to them hasgrown rapidly. Today there are a considerable

number of inherited ion channel diseasesnamed collectively channelopathies, causedby mutations in K+, Na+, Ca2+, and Cl- channelsthat are known to exist in human and animalmodels.

Ion channels constitute a class of macromo-lecular protein tunnels that span the lipidbilayer of the cell membrane, which allow ionsto flow in or out of the cell in a very eYcientfashion (up to 106 per second). This flow ofions creates electrical currents (in the order of10-12 to 10-10 amperes per channel) large enoughto produce rapid changes in the transmem-brane voltage, which is the electrical potentialdiVerence between the cell interior and exte-rior. Inasmuch as Na+ and Ca2+ ions are athigher concentrations extracellularly than in-tracellularly, openings of Na+ and Ca2+ chan-nels cause these cations to enter the cell anddepolarise the membrane. In contrast, when K+

leaves or Cl- enters the cell through open chan-nels, the cell interior becomes more negative,or hyperpolarised. Ion channels in general canbe either open or closed. The process of transi-tion from the open to the closed state (and viceversa) is known as gating. Some channels openand close randomly at all membrane potentials.Their gating is said to be voltage independent.Other ion channels are normally closed buttheir open probability can be greatly enhancedby a change in membrane potential (voltagegated channels), by the binding of extracellularor intracellular ligands (ligand gated channels),or by physical stimuli (mechano and heat sen-sitive channels). When an ion channel opens,permeant ions are able to move through it, andthe direction in which they move, as mentionedabove, is governed by the electrochemicalgradient that represents the sum of the chemi-cal gradient across the plasma membrane andthe electrical field experienced by the ion.Nevertheless, the movement of an ion throughan open channel is not only a function of itselectrochemical gradient. It is also dependenton the relative permeability of the channel tothe ion, which is determined by several factorsincluding the relative sizes of the ion and thepore of the channel. However, ion channels donot act only as molecular sieves that allow thefree diVusion of ions below a certain size.Rather, they can discriminate in the kind ofions to which they are permeable. For example,

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Department ofPhysiology,Biophysics, andNeuroscience, Centerfor Research andAdvanced Studies ofthe NationalPolytechnic Institute(Cinvestav-IPN),MexicoR Felix

Correspondence to:Dr Felix, Departmento deFisiologa Biofsica yNeurociencias,Cinvestav-IPN, Avenida IPN2508, Colonia Zacatenco,Mxico DF, CP 07000,rfelix@fisio.cinvestav.mx

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Na+ channels are highly permeable to Na+ butnot to K+ ions, whereas K+ channels are 100times more permeable to K+ than to Na+.Because Na+ has a smaller ionic radius than K+,the high selectivity of the channels cannot besimply explained by physical occlusion. Thisionic discrimination takes place where the poreof the channel is narrowest, at a region knownas the selectivity filter, and it is the amino acidslocated at the selectivity filter that determinewhich ions can permeate. Cation selectivechannels, for example, often have negativelycharged residues at, or near, their selectivity fil-ters, which attract positive ions and repel nega-tive ions.1 2

This review focuses on the voltage gated ionchannels for cations (Na+, K+, and Ca2+), andbriefly discusses several recent studies linking agrowing number of genetic disorders to genesof the ion channel superfamily, with specialemphasis on those characterised by neurologi-cal, neuromuscular, or cardiac dysfunction inhumans and mice. However, in order to be ableto put the information on channelopathies inperspective, it is necessary to consider firstsome molecular aspects of voltage gated ionchannel structure and function.

Voltage gated ion channels and cellularexcitabilityVoltage gated ion channels can assume either ofthree major conformational states, closed,open, or inactivated, and usually respond tomembrane depolarisation with a transitionfrom the closed to an open state followed by anintrinsic inactivation (fig 1A).1 2 Na+ and K+

channel activation and inactivation are thebasis of the action potential, the principal inte-grating signal that allows excitable cells to con-duct information for the control of a widerange of physiological events including, amongothers, propagation of the nerve impulse andcardiac pacemaking (fig 1B). Furthermore,Ca2+ channels not only contribute to mem-brane polarisation per se, but also play an addi-tional critical role in controlling the influx ofthis important intracellular regulator. Oncewithin the cell, Ca2+ interacts with modifyingenzymes, cofactors, and other second messen-gers to influence cellular events ranging frommuscle contraction, exocytosis, and enzymeactivation to gene expression.2 3 Voltage gatedion channels may also participate in the regula-tion of many specific functions in non-excitablecells. For example, in mammalian spermcapacitation, a poorly understood maturationalprocess, a modification of K+ channel activity,occurs that render sperm responsive to stimulileading to the acrosome reaction beforefertilisation.4

Molecular structure of voltage gated ionchannelsAs mentioned above, ion channels have distinction selectivity and gating properties, deter-mined at the molecular level by the type of thepore forming () subunit that the channel con-tains (fig 2A). The structure of the subunits ismodelled as six hydrophobic segments (S1-S6)embedded in the plasma membrane with the

N- and C-terminal domains of the proteinpositioned intracellularly. One transmembranesegment (S4) contains a unique array ofpositive charges that function as the voltagesensor of the channel. The region separatingsegments S5 and S6 may contain two segmentsthat together form the pore of the channel.1 2

Figure 1 Ion channels and electrical excitability. (A)Plasma membrane voltage gated channels can adopt atleast three conformational states. At rest, the closed (but notinactivated) state has the lowest free energy and is thereforemost stable; however if the membrane is depolarised, theenergy of the open state is lower and the channel opensallowing ions to flow. The cell is saved from a permanentspasm because the free energy of the inactive state is lowerstill, and after a randomly variable period spent in the openstate the channel becomes inactivated (stops conducting). Inthis state the channel cannot open again until themembrane potential returns to its initial negative value.(B) Voltage gated ion channels are responsible forgenerating transient self-propagating electrical signals calledaction potentials. The time course of a prolonged cardiacaction potential is illustrated. In this particular case, anexcitatory stimulus that causes a momentary partialdepolarisation beyond a threshold voltage promptly causesvoltage gated Na+ channels to open (1) causing furtherdepolarisation of the cell membrane.Na+ channels are thenrapidly inactivated allowing a transient K+ current toreturn the action potential to a plateau voltage (2). Thisplateau is maintained by the balance between outward andinward moving currents through K+ and Ca2+ channels,respectively. Progressive inactivation of Ca2+ channels andincreasing activation of K+ channels repolarise the cell (3)to the resting membrane potential (4), which is maintainedby inwardly rectifying K+ channels.

50

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Figure 2 Structure and function of voltage gated K+ channels. (A) Voltage gated K+ channels of the Shaker related superfamily are assembled from membraneintegrated subunits and auxiliary subunits. The subunits may increase K+ channel surface expression and confer native kinetic behaviour to subunit K+channels expressed in heterologous systems. (B) A functional voltage gated K+ channel is formed by four subunits clustered around the central pore.Atetrameric channel complex can be formed by physical association of identical (homomers) or diVerent (heteromers) subunits. (C) The most recently discoveredfamily of ion channels is that containing the inwardly rectifying K+ selective channels (IRKs).The general structure of an IRK channel consists of twomembrane spanning domains (M1 and M2) that flank a highly conserved pore (P) region containing the conserved H5 segment (upper panel). The H5 andM2 segments, in conjunction with the carboxyl-terminus hydrophilic domain, are critical for K+ permeation.As in Shaker-like channels, four channel subunitspresumably assemble to form a functional IRK channel. This group of K+ channels conducts current (IK) much more eVectively into the cell than out of it anddetermine the transmembrane voltage (Vm) of most cells at rest (see also fig 1B), because they are open in the steady state (lower panel). (D) Using positionalcloning methods to establish a gene, it was possible to identify the gene (KVLQT1) responsible for long QT syndrome.KVLQT1 is strongly expressed in theheart and encodes a protein with structural features of a voltage gated K+ channel. (E) The voltage gated K+ channel KCNQ1 associates with the KCNE1subunit (a small one transmembrane segment protein, right panel) to form the slow cardiac outward delayed rectifier K+ current (IKs).KCNQ1 can formfunctional homotetrameric channels, but with drastically diVerent biophysical properties compared to heteromeric KCNQ1/KCNE1 channels.A great deal ofunderstanding of cardiac arrhythmogenesis is emerging as the function of these channels has been elucidated. In fact,mutations in both subunits can cause longQT syndrome (listed in table 1). (F) The HERG K+ channel is atypical in that it seems to have the structure of the voltage gated K+ channel family (sixputative transmembrane segments), yet it exhibits rectification like that of the inward rectifying K+ channels, a family with diVerent molecular structure (twotransmembrane segments, illustrated in C).Functional studies on HERG channels have suggested that the inward rectification arises from a rapid and voltagedependent inactivation process that reduces conductance at positive voltages.

Voltage gated (Shaker-like)

Pore

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COOH

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+S5 S6

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COOH

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KCNE1

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COOH

Vm (mV)0

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Most of the functional K+ channels areformed when four subunits aggregate to pro-duce a hetero- or homotetrameric structure (fig2B). However, evidence exists that some K+

channels more distantly related to this super-family of voltage gated channels (for example,the inwardly rectifying and the G protein regu-lated inwardly rectifying K+ channels, IRKsand GIRKs, respectively) possess only twomembrane spanning segments that resemblethe S5 and S6 regions of the subunit (fig 2C).Functional Na+ and Ca2+ channel subunitscontain four sets of S1-S6 membrane spanningregions coded as parts of a single gene productlinked by hydrophilic loops. Even though the subunits are capable of conducting ions bythemselves, voltage gated ion channels ofteninclude one or more ancillary proteins calledauxiliary subunits that play modulatory,structural, or stabilising roles.5 These auxiliarysubunits can be subdivided into two mainclasses. One class consists of the entirely cyto-plasmic intracellular subunits, which have notransmembrane domains such as the subu-nits of the voltage gated K+ (fig 2A) and Ca2+

channels (fig 3B). The other class of auxiliarysubunits contains at least one transmembranedomain. These proteins include the 2 and subunits of the Ca2+ channel (fig 3B) and the subunits of the Na+ channel (fig 3A).6 Mosttraditional voltage gated K+ channels have notbeen shown to have transmembrane containingsubunits, although a family of membraneproteins including IsK (minK) may fit into thiscategory (fig 2E).

Human channelopathies and mousemodelsK

+CHANNELOPATHIES

Episodic ataxia type 1 (EA1) is the only humanataxia known to be caused by dysfunction of aK+ channel (table 1). Some other types of thisneurological disorder are associated with muta-tions in Ca2+ channels (see below). EA1 is arare, disabling condition of autosomal domi-nant inheritance. Although family membershave similar clinical features, the syndromevaries considerably from family to family. Thedisorder begins in early childhood with attacksof ataxia of one to two minutes in duration,with associated interictal uncoordinated move-ments of the head, arms, and legs. Attacks areprovoked by abrupt postural change, emotionalstimulus, and caloric vestibular stimulation.EA1 is the result of diVerent heterozygous mis-sense point mutations in a neuronal voltagegated (delayed rectifier) K+ channel gene

(KCNA1/Kv1.1) on chromosome 12p13 (table1).7 Expression in Xenopus oocytes showed thattwo of these EA1 subunits are indeed able toform functional channels, though their gatingproperties are altered. A Val408 to Alamutation produces channels with voltagedependence similar to that of wild typechannels, but with faster kinetics and increasedC type inactivation (a slow inhibition in chan-nel activity induced by voltage), while the volt-age dependence of Phe184 to Cys mutantchannels is shifted 20 mV in the depolarisingdirection. Several other EA1 subunits havefailed to produce functional homomeric chan-nels, but reduced the K+ current whencoassembled with wild type subunits.8 Becausestudies in Xenopus oocytes indicate that themutations causing EA1 result in reductions inKCNA1 expression and current amplitude,knockout mice lacking the murine homologueof KCNA1 became a useful animal model forEA1. Mice heterozygous for the null alleleappear normal while homozygous mutantshave very frequent generalised epilepticseizures.9

Extensive further work has provided usefulclues for understanding the basis of the EA1phenotype. By using specific antibodies, it wasfound that kcna1 is localised to a key site regu-lating cerebellar output, the axons and nerveterminals of the basket cells that makeinhibitory synaptic contacts on Purkinjeneurones.10 Patch-clamp recordings of thesecells showed an extraordinarily high K+ chan-nel density. Blocking these channels with-dendrotoxin caused a dramatic increase inthe spontaneous inhibitory postsynaptic poten-tials recorded in Purkinje cells, suggesting thatkcna1 regulates the excitability of the basketcell presynaptic terminals.11

To date, all inherited arrhythmogenic disor-ders in which the causative genes have beenidentified turned out to be channelopathies,since the genes encode channel subunits thatregulate important ion currents that tune thecardiac action potential. Congenital long QTsyndrome was the first genetically determinedarrhythmia shown to be caused by defects inmyocardial ion channels. This clinical condi-tion involves an alteration characterised byprolongation of the QT interval of the electro-cardiogram, which results from an increasedduration of the cardiac action potential. Mostpatients suVering from long QT syndrome areasymptomatic and only one third of the casesare identified by electrocardiographic screen-ing during a clinical evaluation for either unex-

Table 1 K+ channel genes associated with neurological and cardiac disorders in human and mouse

K+ channel gene Human disease Mouse model Common mutations

KCNA1 Episodic ataxia type 1 V408A; R239S; V174F; F249I; F184C; E325D; T226A; V404I; I176RKCNE1 Long QT syndrome type 1 S74L, D76NKCNQ1 Long QT syndrome type 1 3 nucleotides deletion (TCG in codon 72); A83P; G189R; R95Q; V159M;

L178F; G211R; T217I; A341E; A341V; G250E; G219S; R555C; W305S;A300T; R518T; A529T; 3 bp deletion (F339); 9 bp deletion del373-381

HERG Long QT syndrome type 2 1 bp deletion (1261); 27 bp deletion (1500); A561V; N470D; I593R; V822M;G628S; R582C

KCNJ6 (GIRK2) Weaver G156SKCNQ2 Benign familial neonatal convulsions Y284C, A306T; 2 bp insertion (283 ins GT); 13 bp deletion at amino acid 522;

1 bp deletion, del2513GKCNQ3 Benign familial neonatal convulsions G263V

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plained syncope or cardiac or respiratoryarrest. Often the arrhythmia is a polymorphicventricular tachycardia known as torsade despointes, triggered by adrenergic arousal or byexposure to a variety of medications.12 13 Recentstudies have implicated several genetic defectsin KCNQ1 (table 1), a 581 amino acid proteinwith sequence homology to the subunit ofvoltage gated K+ channels (fig 2D) in the aeti-ology of the disease.1416

The association of this KCNQ1 channelprotein with another subunit named IsK (alsoknown as minK) initiates the IKs current (fig

2E), which is the principal delayed rectifyingK+ current responsible for repolarising the car-diac myocytes during the action potential.17

Mutations in the K+ channel gene (KCNQ1)and the consequent alteration of the IKs currentresults in sustained depolarisation and abnor-mally long cardiac action potentials. Similarly,physiological studies showed that two muta-tions in the KCNE1 gene encoding the humanminK subunit significantly reduced IKs currentby altering some of the fundamental biophysi-cal properties of the channel including its volt-age dependence of activation and kinetics of

Figure 3 Subunit structure of the voltage gated Na+ and Ca2+ channels. (A) Na+ channels are responsible for actionpotential generation in most excitable cells and consist of a complex of three glycoprotein subunits: a pore forming subunitof 260 kDa associated non-covalently with a 1 subunit of 36 kDa and disulphide linked to a 2 subunit of 33 kDa.The subunit forms a functional channel by itself, and is composed of four homologous domains (I-IV), which contain sixprobable helical transmembrane segments (S1-S6) and an additional membrane associated pore loop. 1 and 2 subunitsare single membrane spanning glycoproteins that modulate channel gating. Structure-function analyses of the subunithave shown that the S4 transmembrane segments in each domain serve as voltage sensors for channel activation, the S5and S6 segments and the pore loop between them form the transmembrane pore, and the highly conserved intracellular loopbetween domains III and IV is proposed to form the inactivation gate (arrow). The auxiliary subunits do not form thepore but play important roles in modulating channel function, as well as plasma membrane expression levels. (B) Ca2+channels provide a crucial link between a cells membrane potential and an enormous number of intracellular processes thateither directly use increases of [Ca2+]I as a functional trigger (for example, exocytosis, muscle contraction) or are modulatedby Ca2+ dependent signalling cascades (for example, gene expression, cell division). These channels are complexes of a poreforming 1 subunit, a transmembrane subunit disulphide linked to an extracellular 2 subunit, an intracellular subunit,and, at least in some tissues, a subunit. Expression of cloned 1 genes has shown many functional roles for this subunit,including sensing of the transmembrane voltage, channel activity, and binding of pharmacological agents. As in voltagegated Na+ channels, the 1 subunit (190-250 kDa) of Ca

2+ channels is composed of four homologous membrane spanninginternal domains, each with six transmembrane helices and a pore forming re-entrant P loop. Carboxyl- and amino-terminiand linkers between domains I-II, II-III, and III-IV are cytosolic. The 2 complex (170 kDa) derives from a commonprecursor protein that is proteolytically processed to yield separate 2 and proteins that remain linked by disulphide bonds.This auxiliary subunit determines membrane localisation and modifies distinct biophysical and pharmacological propertiesof the ion conducting 1 subunit. The ancillary subunits are cytoplasmic proteins (of 50-80 kDa), which facilitate theincorporation of channels into the plasma membrane and modulate Ca2+ currents. The subunit is a 26 000 daltonglycosylated hydrophobic protein that is predicted to contain four transmembrane domains. Functional studies suggest that is important in channel assembly and modulates channel properties in a subtype specific manner.

HOOC COOH

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I II III IV

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+S5 S6

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S1 S2 S3 S4+

+S5 S6 S1 S2 S3 S4

+

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+

+S5 S6

NH2

HOOCH2N

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inactivation.18 The main functional conse-quence of these mutations would be delayedcardiac repolarisation and an increased risk ofarrhythmia.

Mutations in a third K+ channel known asHERG (human ether-a-go-go-related gene, fig2F) have been identified in subjects suVeringfrom a second form of the long QT syndrome(type 2). HERG is responsible for anothermajor K+ current IKr that participates in therepolarisation of the cardiac action potential.Although HERG has the secondary structureof a typical voltage gated K+ channel (fig 2A),it behaves like an inward rectifier because itconducts current much more eVectively intothe cell than out of it. The role of this channelin cardiac physiology appears to be to suppressdepolarisations that lead to premature firing.Patients with long QT syndrome type 2 maytherefore be prone to sudden cardiac death,because they lack protection from arrhyth-mogenic afterbeats.19 20

The discovery of the genetic bases of theLQT syndrome became a new methodologicalparadigm. With the use of genetic linkagestrategies, not only have the causative genesbeen found, but functional components with apreviously unknown but fundamental role for anormal repolarisation process have been dis-covered.

Two rare forms of epilepsy known as benignfamilial neonatal convulsions (BFNC) type 1and 2 have been linked to mutations in twonovel and related genes (KCNQ2 and KCNQ3,respectively) coding for the voltage gated K+

channel subunit (table 1). These forms ofepilepsy are characterised by frequent, briefseizures after the second day of life which oftendisappear spontaneously within weeks. New-borns show normal behaviour between seizuresand normal subsequent neurodevelopment.The seizure phenotype usually starts with atonic posture and shallow breathing, followedby ocular signs (staring, blinking, or gazedeviation), clonic movements, and automa-tisms. Electroencephalograms recorded duringseizures show a characteristic sequence ofalterations that correlate with clinical signs. Inthe case of BFNC1, the genetic defects includetwo missense, two frameshift, and one splicesite mutations, while in patients with BFNC2the alteration comprises a single missensemutation in the pore region of the channel(table 1). Since voltage gated K+ channels areresponsible for repolarising the cell membraneduring the action potential, and they are alsothought to participate in membrane repolarisa-tion after activation of excitatory neurotrans-mitter ion channels, it has been speculated thatin the presence of mutant K+ channels withreduced function, both voltage and ligandgated ion channels would remain openlonger.21 22 Recently, Wang et al23 reported thatheterotetramers consisting of KCNQ2 andKCNQ3 form the so called M channels thatregulate the subthreshold electrical excitabilityof neurones. Although it is likely that BFNC iscaused by neural hyperexcitability owing todysfunction of M channels, there has been nodirect evidence that an alteration in a KCNQ

gene can actually cause epilepsy.23 However,mice heterozygous for the null allele of theKCNQ2 gene showed normal behaviour andmorphology compared with wild type mice, butdisplayed increased sensitivity to an epilepticinducer.24

Lastly, two more targeted mutations in themurine orthologues of the subunit of K+

channels have been described. Their pheno-types vary from seizures to more subtle signs,including poorly coordinated motor skills andinner ear defects.25 26 Perhaps the best studiedK+ channel linked disorder in mouse is theweaver (wv) mutation, which results from agenetic defect in the KCNJ6 gene encoding theG protein coupled inwardly rectifying K+

channel (GIRK2).27 28 Weaver mice are charac-terised by male infertility, spontaneous sei-zures, extensive cerebellar granule cell death,and progressive loss of substantia nigra neu-rones. Neuronal death in weaver has also beenattributed to the loss of G protein activatedinwardly rectifying K+ currents in animalscarrying the mutant allele.29 Because of thepostnatal degeneration of dopaminergic neu-rones exhibited by wv/wv mutant mice, weaveralso provides an interesting animal model forParkinson disease.

NA+

CHANNELOPATHIES

The periodic paralytic disorders of skeletalmuscle and the non-dystrophic myotonias arediseases resulting from mutations in voltagegated Na+ channels (fig 3A). In particular,hyperkalaemic periodic paralysis (HyperPP),paramyotonia congenita (PMC), acetazola-mide responsive myotonia, and myotoniapermanens/fluctuans are all clinically distinctautosomal dominant disorders associated withmutations in the subunit of Na+ channels.

HyperPP is an autosomal dominant disordercharacterised by episodes of muscle weaknessresulting from depolarisation of the muscle cellmembrane associated with raised serum K+.Early molecular studies found that the adultmuscle Na+ channel subunit gene (SCN4A)contained the HyperPP mutation.30 Analysis ofDNA from unrelated patients with HyperPPshowed a C to T transition, predicting a substi-tution of a highly conserved threonine residuewith a methionine in a membrane spanningsegment of the Na+ channel protein.31 Furthergenetic analysis of the subunit coding regionfrom aVected HyperPP patients identified sev-eral mutations that cause changes in both thehighly conserved transmembrane regions andthe variable cytoplasmic domains of the Na+

channel subunit (table 2). Electrophysiologi-cal data indicate that Na+ channels from mus-cle cells of HyperPP aVected subjects showabnormal activation32 and inactivation.33 34

Similarly, several mutations that occur in theadult skeletal muscle voltage gated Na+ channelSCN4A gene (table 2) have been identified ascausing paramyotonia congenita (PMC), atemperature sensitive disorder characterised byexercise and cold induced myotonia and weak-ness. Recent studies have emphasised the roleof channel mutations that cause a positivecharge decrease in segment S4 of domain IV of

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the ion conducting subunit. In particular, asingle nucleotide substitution causing anamino acid change at position 1448 in segmentS4 of domain IV has been implicated in thephenotype of PMC. Interestingly, expression ofthe altered proteins in HEK293 cells showedseveral defects in channel function includingalterations in the kinetics and voltage depend-ence of inactivation, slower deactivation, andfaster recovery from inactivation.3538

Ricker et al39 discovered a novel mutation inexon 22 of the Na+ channel SCNA4 gene,which encodes the region containing the cyto-plasmic loop between domains III and IV. A Gto C transition was found at position 3917,predicting a Gly1306 to Ala substitution. Thismutation was found in aVected members offamilies with the myotonia fluctuansphenotype.39 This consists of fluctuating myo-tonia of varying severity, a warm up phenom-enon, worsening of myotonia after K+ loading,increased myotonia with delayed onset follow-ing exercise, and no significant increase ofmyotonia following exposure to cold. Similarly,one missense mutation (I1160V) that occurs ata very highly conserved position in the Na+

channel subunit has been reported as theputative disease causing mutation in acetazola-mide responsive myotonia congenita,40 41 anautosomal dominant, painful myotonia that isK+ sensitive and responsive to acetazolamide.

Myotonic muscles recorded in vivo exhibitabnormal spontaneous oscillations in mem-brane potential, called myotonic discharges. Innormal muscle, depolarisation of the postsyn-aptic membrane causes the opening of Na+

channels within the first few msec afterdepolarisation. As these Na+ channel openingssubside, Cl- enters the cell through more slowlyopening Cl- channels, promptly returning themembrane potential to its resting level. Na+

channels harbouring mutations causing myoto-nia exhibit an abnormal tendency to open lateror more persistently after membrane depolari-sation. Residual Na+ entry through theseabnormal channels repeatedly reinitiates thecycle of membrane depolarisation.42

As mentioned above, heritable long QT syn-drome is a disease in which delayed ventricularrepolarisation leads to cardiac arrhythmias andthe possibility of sudden death. Wang et al23

mapped three LQT loci: LQT1 on chromo-some 11p15.5, LQT2 on 7q35-36, and LQT3on 3p21-24. They reported genetic linkagebetween LQT3 and polymorphisms withinSCN5A, the cardiac Na+ channel gene. Singlestrand conformation polymorphism and DNAsequence analyses showed intragenic deletions

of SCN5A in aVected LQT patients, indicatingthat mutations in SCN5A cause chromosome 3linked LQT.43 In the disease, one mutation ofthe cardiac Na+ channel subunit gene resultsin a deletion of residues 1505 to 1507, and twomutations result in substitutions (N1325S andR1644H). These altered sequences reside in aregion that is important for channel inactiva-tion, suggesting a likely cellular mechanism forthe cardiac disorder. LQT3 mutant channelexpression in Xenopus oocytes produced inacti-vation resistant whole cell currents.4446

A transgene induced intragenic deletion ofthe brain Na+ channel Scn8a has beenidentified in mice with motor end plate disease(med), a genetic defect that causes progressiveneuromuscular failure. Mice homozygous forthis mutation display altered cerebellar func-tion and progressive neuromuscular weakness,which begins 10 days after birth and results indeath within 3-4 weeks.47 Analysis of homo-zygous med mice showed that the developmentand function of mature motor neuronesdepends on the postnatal induction of Scn8aexpression.48 Similarly, Kohrman et al49 showedthat a mutation that shifts the voltage depend-ence of activation of the channel is associatedwith inherited cerebellar ataxia in the joltingmouse.49 This mutant mouse arose as aspontaneous mutation and genetic mappingand complementation tests showed that joltingis a mild allele (medjo) of the motor endplatedisease locus (med) on mouse chromosome 15that exhibits cerebellar ataxia only.50 Thejolting mutation results in substitution of Thrfor a conserved Ala residue in the cytoplasmicS4-S5 linker of domain III in the subunit ofthe channel (fig 3A). Homozygous jolting micehave ataxia of cerebellar origin and a rhythmi-cal tremor of head and neck. Electrophysiologi-cal studies have shown that the absence ofspontaneous, regular, simple discharges fromthe Purkinje cells in the cerebellum of medjo

mice, and the introduction of the jolting muta-tion into the rat brain IIA Na+ channel,expressed in Xenopus oocytes, shifted the volt-age dependence of activation in the depolaris-ing direction, providing the molecular basis forthe reduced spontaneous activity of Purkinjecells, reduced inhibitory output from thecerebellum, and loss of motor control observedin the jolting mouse.51

CA2+

CHANNELOPATHIES

Molecular biological analyses have identified agene family of 1 subunits (A, B, C, D, E, F, G,H, I, and S) encoding six functionally diVerenttypes (L, N, P, Q, R, and T) of Ca2+ channels(fig 3B). OphoV et al52 identified mutations in

Table 2 Na+ channel genes associated with neurological and cardiac disorders in human and mouse

Na+ channel gene Human disease Mouse model Common mutations

SCN4A Hyperkalaemic periodic paralysis T698M; T704M; M1595V; M1592V; V783I; M1360VSCN4A Myotonia fluctuans G1306ASCN4A Myotonia permanens G1306ESCN4A Myotonia acetazolamide responsive I1160VSCN4A Paramyotonia congenita I693T; V1293I; G1306V; T1313M; L1433R; R1448C; R1448H,

R1448P; R1448S; V1458F; F1473S; V1589MSCN5A Long QT syndrome type 3 Deletion KPQ 1505-1507; N1325S; R1644H; R1623QScn8a Motor end plate disease Transgene induced intragenic deletionScn8a Jolting A1071T

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the human Ca2+ channel 1A subunit CACN1A4gene in episodic ataxia type 2 (EA2) and famil-ial hemiplegic migraine (FHM) patients. EA2is an autosomal dominant paroxysmal cerebraldisorder, characterised by acetazolamide re-sponsive attacks of ataxia and migraine-likesymptoms, interictal nystagmus, and cerebellaratrophy. FHM is a rare autosomal dominantsubtype of migraine with aura, associated withictal hemiparesis and, in some cases, progres-sive cerebellar atrophy. In EA2, two mutationsdisrupting the reading frame were originallyfound, one base pair deletion (C4073) result-ing in a truncated Ca2+ channel 1 subunit anda G to A transition of the first nucleotide ofintron 24 (table 3). Four diVerent missensemutations were identified in FHM patients: atransition from G to A at codon 192, leading tosubstitution of an Arg for Glu (R192Q) withinthe fourth segment of the first membranespanning domain (IS4); a missense mutation atthe second repeat, replacing a Thr with Met(T666M); and two mutations located in thesixth transmembrane spanning segment ofrepeats II and IV (V714A and I1811L, respec-tively). Functional studies provided evidencethat three of these four mutations aVect thekinetic properties and the voltage dependenceof activation of the 1A Ca

2+ channel.53 In addi-tion, a locus for spinocerebellar ataxia type 6(SCA6), which is another autosomal dominantparoxysmal cerebral disorder, was mapped tochromosome 19p13.1 in the same interval asthe EA2 and FHM locus,54 55 indicating thatEA2, FHM, and SCA6 are allelic Ca2+ channeldisorders. Histological studies showed neuro-nal degeneration confined mainly to thecerebellar Purkinje cells,56 suggesting a likelypathological mechanism for the neurologicalphenotype. Interestingly enough, a polymor-phic CAG repeat was identified in the humanCa2+ channel 1A subunit,

57 58 providing themolecular basis for this disorder.

Incomplete X linked congenital stationarynight blindness (CSNB2) is a recessive non-progressive retinal disorder characterised bynight blindness, decreased visual acuity, myo-pia, nystagmus, and strabismus. Electrophysi-ological data suggested a defect in retinal neu-rotransmission, while molecular studieslocalised the CSNB2 locus to chromosomeXp11.23.59 Interestingly, a Ca2+ channel 1

subunit gene (CACNA1F), which shares highhomology with the dihydropyridine (DHP)sensitive L type channel was identified in thisregion.60 61 Genetic analysis of this novel genein CSNB2 patients showed several diVerentmutations (table 3), including nonsense andframeshift mutations that would be predictedto cause premature protein truncation, suggest-ing that aberrations in this voltage gated Ca2+

channel cause this disorder.5961

Hypokalaemic periodic paralysis (HypoPP)is an autosomal dominant muscle diseasemanifested by episodic weakness associatedwith low serum K+. It is thought to arise alsofrom the abnormal function of Ca2+ channels.Fontaine et al62 localised the HypoPP locus tochromosome 1q31-32 and showed that thegene encoding the skeletal muscle DHP sensi-tive Ca2+ channel 1S subunit (CACN1A3)maps to the same region. Ptacek et al63 showedthat mutations in the CACN1A3 gene causing asubstitution of a highly conserved Arg in the S4segment of domain IV in 1S with either His orGly are associated with HypoPP. Functionalexpression of these mutant subunits, as well asa third mutation in the S4 segment of domainII (R528H), in Xenopus oocytes showedreduced L type Ca2+ current amplitude andaltered activation properties.64 Although thismutation induced only minor diVerences in theelectrophysiological properties of 1S, it signifi-cantly reduced the whole cell Ca2+ current in itsamplitude when expressed in mouse Ltk-cells.65

Initial studies in subjects suVering frommalignant hyperthermia susceptibility (MHS),a potentially fatal autosomal dominant disor-der of skeletal muscle triggered in susceptiblepeople by inhalation anaesthetics, indicatedthat a mutation in an intracellular Ca2+ releas-ing channel, the ryanodine receptor type 1(RYR1), was linked to the disease.66 However,the demonstration of genetic heterogeneity inMHS67 prompted the investigation of the rolesplayed by Ca2+ regulatory proteins other thanRYR1, which was known to be linked to MHSin fewer than half of the cases.68 In severalMHS families, Iles et al69 found linkage with norecombination to markers flanking theCACN2A locus on chromosome 7. Since thisgene encodes the 2 subunit of the L typevoltage gated Ca2+ channel that is intimately

Table 3 Ca2+ channel genes associated with neurological and neuromuscular disorders in human and mouse

Ca2+ channel gene (formernomenclature)

Ca2+ channel gene(HUGO/GDB genenomenclature)96 97 Human disease Mouse model Common mutations

CACNL1A4 (1A) CACNA1A Episodic ataxia type 2 1 bp deletion (del4073C); IVS24DS, G-A, +1;(CAG)n expansion

CACNL1A4 (1A) CACNA1A Familial hemiplegic migraine R192Q; T666M; V714A; I1819L; D715ECACNL1A4 (1A) CACNA1A Spinocerebellar ataxia type 6 (CAG)n expansion; G293RCACNL1A4 (1A) CACNA1A Tottering P647LCACNL1A4 (1A) CACNA1A Leaner Splicing errorCACNL1F (1F) CACNA1F Incomplete X linked congenital

stationary night blindnessG369D, R508Q, R1049W, L1364H;1 bp insertion (3133insC);12 bp deletion (del3658-3669);3 stop codon causing mutations (R958X,Q1348X, K1591X)

CACNL1A3 (1S) CACNA1S Hypokalaemic periodic paralysis R528H; R1239H; R1239GCACNL1A3 (1S) CACNA1S Malignant hyperthermia susceptibility R1086HCACNLA2 (2) CACNA2D1 Malignant hyperthermia susceptibility UnknownCACNLB4 (4) CACNAB4 Lethargic Splicing errorCACNLG2 (2) CACNG2 Stargazer Early transposon insertion

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associated at the skeletal muscle triadic junc-tions with the RYR1, it was thought that themutation was located in this gene. This, andthe recently discovered mutations in the 4subunit gene (see below) in aVected subjectswith idiopathic generalised epilepsy and he-reditary episodic ataxia, are the only directimplication of an auxiliary Ca2+ channel protein(fig 3B) in human genetic disease.70 Althoughdetailed analysis of the sequence and genomicstructure of the CACNA2 gene have been per-formed, mutations within the coding region ofthis gene have not yet been identified.71

However, since the promoter region remains tobe analysed, and the eventuality of an intronicmutation is still possible, CACNA2 remains aprime candidate for MHS in subjects whoshow no mutation in the Ca2+ channel ion con-ducting subunits of skeletal muscle. Howmutations in either RYR1 or the Ca2+ channel2 subunit might result in the same diseaseremains unknown. However, both Ca2+ chan-nels are involved in excitation-contraction cou-pling and have been shown by electron micros-copy to be in close approximation to each otherin skeletal muscle.72 Thus, the possibility existsthat mutations in the 2 subunit couldinfluence the behaviour of the 1S subunit as avoltage sensor for excitation-contraction cou-pling. More recently, evidence suggesting twofurther loci for MHS was published.73 One ofthese is located in chromosome 1q, the site of acandidate gene, CACN1A3, encoding the 1Ssubunit of the DHP sensitive skeletal muscle Ltype Ca2+ channel. The second region resideson chromosome 5p where no known candidatehas been mapped yet. Sequence analysis of thecoding region of the CACN1A3 gene showedthe presence of an Arg-His substitution at resi-due 1086, resulting from the transition of A toG3333, which segregates with the MHSphenotype.74

As mentioned above, very recently twomutations have been reported in the human 4subunit gene (R482X which results in a proteintruncated in the middle of the domain thatinteracts with the C-terminus of the 1 subunit,and a GT transversion resulting in the replace-ment of a Cys residue 104 by Phe) cosegregatewith the disease in aVected subjects suVeringfrom idiopathic generalised epilepsy and epi-sodic ataxia.70 However, the alterations of the1A Ca

2+ currents caused by the functionalexpression of these mutant subunits in theoocyte expression system were too small tosupport linkage.70

Similarly, various spontaneous mutants andnatural strain variants for either generalisedtonic-clonic seizures or non-convulsive ab-sence seizures have been described in mice overthe years. Some of these abnormalities havebeen attributed to specific alterations of Ca2+

channel activity. Hence, a missense mutationwas found in the voltage gated Ca2+ channel 1Agene in the tottering (tg) mice, which display adelayed onset, recessive disorder consisting ofataxia, motor seizure, and absence seizureresembling petit mal epilepsy. The tg mutationcauses a Leu-Pro substitution at a positionclose to the conserved pore lining region (P

region) in the extracellular segment of repeatII. Mice with an allelic tottering mutationleaner (tgla), which causes more severe symp-toms, were found to have a single nucleotidesubstitution at an exon/intron junction, whichresults in skipping of the exon, or results infailure to splice out the succeeding intron. Inboth cases, the tgla mutation causes truncationof the normal open reading frame and expres-sion of aberrant C-terminal sequences.75 Com-parison of the properties of Ca2+ channelcurrent in Purkinje cells of normal andtottering mutant mice showed a reduction ofcurrent density76 77 and changes in channel gat-ing, which could be reproduced with recom-binant mutant channels expressed in a mam-malian cell line.78 A third additional totteringmutation, found in rolling Nagoya (tgrol) mice,manifests with poor motor coordination lead-ing to falling and rolling, and in some casesstiVness of the hindlimbs and tail, but no motorseizures. Recently, Mori et al79 found that thetgrol mutation leads to a charge neutralising Argto Pro substitution in the voltage sensor form-ing segment S4 in repeat III of the 1A subunit.Electrophysiological analysis of 1A Ca

2+ chan-nels in tgrol mutant mouse indicated decreasedcurrents and deviation in the voltage sensingmechanisms of the channels.80

In addition to these mutations in the 1A poreforming subunit of the P/Q type Ca2+ channels,alterations in three mouse models have beenassociated with mutations in Ca2+ channel aux-iliary subunits. The mouse mutant lethargic(lh) exhibits severe neurological defects includ-ing ataxia, episodic dyskinesia, and generalisedspike-wave epilepsy owing to a mutation thatdeletes the 1 subunit interaction domain of the4 subunit. Specifically, the lh mutation causesa 4 bp insertion into a splice donor site withinthe Cacnb4 gene on chromosome 2.81 Themutation results in aberrant pre-mRNA splic-ing and translational frameshift and is pre-dicted to encode a severely truncated 4 proteinmissing 60% of the C-terminus relative to wildtype including, as mentioned above, the essen-tial 1- interaction domain. Notably, sub-units expressed with deletions of this domainlose their ability to modulate 1 subunitfunction in vitro.82 More recently, it has beendocumented that neither full length nortruncated 4 protein are expressed as a result ofthe lh mutation.83

While attempting to identify a new humantumour suppressor gene in chromosome region3p21.3, Gao et al84 identified a novel gene thatencodes a homologue of the 2 subunit of Ca

2+

channels (CACNA2D2). Northern analysisshowed that the CACNA2D2 gene is wellexpressed in brain, and in vitro studies showedan increased number of functional channels atthe plasma membrane after cRNA co-injectionwith 1B recombinant Ca

2+ channels in theXenopus oocyte expression system.84 Interest-ingly enough, the human orthologue of thisnovel Ca2+ channel 2 auxiliary subunit hasbeen shown to map to the ducky (du) candidateregion of mouse chromosome 9.84 85 du is aspontaneous autosomal recessive mutation thatis thought to be a valid model of human

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idiopathic generalised epilepsy. Homozygousdu mice also display ataxia, hind brain dysgen-esis and demyelination, and axonal dystrophyof selected nerve fibres.86 Lastly, in the thirdmouse model, the stargazer (stg) mutationcauses absence seizures that are more pro-longed and frequent than any other petit malmouse model. stg mice also have an ataxic gaitand vestibular problems, including a distinctivehead tossing motion. The stargazer locus wasmapped between D15Mit30 and the parvalbu-min gene, and a novel gene whose expression isdisrupted in two stargazer alleles wascharacterised.87 88 This gene, Cacng2, wasshown to encode the first of several subunitsrecently discovered in the nervous system.8891

Transient transfection of 2 in BHK cells stablyexpressing recombinant 1A (P/Q type) Ca

2+

channels decreased the availability of the chan-nels, as indicated by a negative shift in theinactivation curve for the channels in the steadystate.89 Inasmuch as P/Q type channels aremajor mediators of neurotransmitter release atthe presynaptic terminals,92 it was speculatedthat 2 would inhibit presynaptic Ca

2+ entry instg mutant mice, causing an inappropriatemodulation of an inwardly rectifying cationiccurrent (Ih) which normally governs excitabilityin thalamic neurones.93 94 In this manner, theupregulation of Ih in stg would be a likelymechanistic link between the mutation and theepileptic phenotype.89 94

ConclusionAlthough biophysical studies of mutant voltagegated ion channels in vitro allow detailedinvestigations of the basic mechanism underly-ing channelopathies, a full understanding ofthese diseases requires knowing the roles thesechannels play in their cellular and systemiccontext. It should be noted that in many casesthe mutations have been identified but themechanism by which the mutation causes theabnormal phenotype is unclear. For example,the molecular basis of the tottering mouse phe-notype was identified as a point mutationwithin the 1A subunit of the P/Q type voltagegated Ca2+ channel.75 Recently, however, it hasbeen noted that L type Ca2+ channels may con-tribute to the generation of the intermittentdystonia observed in these mice. Even more,the susceptibility of L type channels to voltagedependent facilitation may promote the abnor-mal motor phenotype in the tg mice.95

I would like to thank Drs A Darszon, T Nishigaki, and C LTrevio for helpful comments on the manuscript, and twoanonymous reviewers for constructive criticism. Work in theauthors laboratory is supported by the National Council forScience and Technology (Conacyt, Mexico) Grant 31735-N.

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