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Potassium Channelopathies
Neuropharmacology,Vol. 36, No. 6, PP.755–762, 1997 @ 1997Elsevier Science Ltd. Ml rights reserved Printed in Great Britain 0028-3908/97$17.00 + 0.00
Pergamon PII: S0028-3908(97)00029-4
Review Potassium Channelopathies M. C. SANGUINETTI* and P. S. SPECTOR Department of Medicine, Division of Cardiologyand Eccles Program in Human Molecular Biology and Genetics, Universityof Utah, Salt Lake City, UT84112, U.S.A. (Accepted 31 December 1996)
summary-l%e molecular diversity of K+-selective channels far exceeds any other group of voltage- or ligand-gatedchannels,reflectingtheir early ancestralorigin.This diversityis mirroredby the broad spectrumof physiological functions subserved by these proteins. Potassium channels modulate the resting potential and action potential duration of neurons, myocytes and endocrine cells and stabilize the membrane potential of excitable and nonexcitablecells. In additionto channeldiversity,differentialcellular expressionof K+channels determinesthe specific electrical responsesto stimuli in a particular cell or tissue. This studyreviews the recent genetic and physiological studies of congenital disorderscaused by mutations in genes encoding K+channels. These include the human disorders of episodic ataxia with rnyokymia, long QT syndrome and Bartter’s syndrome,and weaver ataxia in mice. An understandingof the molecularbasis of these diseasescould facilitate the discovery and development of specific pharmacologicaltherapies. 01997 Elsevier Science Ltd. Keywords—Episodic ataxia, long QT syndrome,Bartters syndrome,weaver.
MUTATIONSIN A NEURALDELAYEDRECTIFIERK+ Litt et al. (1994) used linkage studies to localize the CHANNEL,KV1.1CAUSEEPISODICATAXIAAND EA-1 gene to chromosome 12p, where three related K+ MYOKYMIA channelgenes (KCNAl, KCNA5, KCNA6) had previously Episodic ataxia (EA) is a rare autosomal dominant been mapped. Mutational analysis of KCNA1 in seven necrologic disorder characterized by intermittent attacks families identified six different missense mutations of generalized ataxia, caused by physical or emotional (V174F, F184C, R239S, F2491,E325D, V408A) in this stress. Two types of EA are recognized based on the gene (Browne et al., 1994, 1995). One additional family length and severity of the attacks. Ataxia lasts for only a with EA-1 was shown to result from another missense few seconds or minutes in EA-1 and is associated with mutation (C677T) in KCNA1 (Comu et al., 1996). continuous motor neuron activity, which causes muscle KCNA1 encodes the Kvl.1 delayed rectifier K+ channel rippling (myokymia) between and during attacks (Van that is expressed in the cerebellum and peripheral Dyke et al., 1975). Ataxia lasts for several hours to nervous system of the rat (Beckh and Pongs, 1990). several days in EA-2 and is associated with nystagmus Because affected members in all families had similar and cerebella atrophy.Linkage analysismapped the EA- symptoms, it was not possible to make phenotype– genotypecorrelations.Browne et al. (1995)hypothesized 2 disease gene to chromosome 19p13, the same region that mutationsin KCNAI would likely decrease function associated with familial hemiplegic migraine (FHM), of the Kvl.1 channels by a dominant negative effect. suggestingthe possibility that EA-2 and FHM are allelic Adelman et al. (1995) studied the physiological disorders.This was confirmedrecently with the discovery consequences of EA-1 associated mutations in KCNAI that both disorders are caused by mutations in CACN- by expressingthe mutant Kvl.1 proteins, either alone or LIA4, a brain-specificP/Q-type Ca2+channel (Ophoff et in combination with wild-type subunits in Xenopus al., 1996). oocytes. Four of the mutant Kvl.1 subunits (V174F, . R239S, F2491, E325D) did not induce expression of *To+whomcorrespondenceshouldbe addressed.Tel: 801-585- functional channels, but suppressed current when coex6336; Fax: 801-585-3501;e-mail: mike.sanguinetti@gene- pressed with wild-type subunits; a dominant negative effect. The R239S and E325D mutant subunits greatly tics.utah.edu 755
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M. C. Sanguinetti and P. S. Spector Table 1. Summary of diseases caused by mutations in K+ channel genes
Disease EA-1 (episodic ataxia and
Mutated K+ channel gene
Coassembles as tetrarner to form neural delayed rectifier K+channel
Missense (V174F,F184C, R239S, F2491,E325D, V408A, C677T)
HERG
Coassembles as tetramer to form rapid delayed rectifier K+ channel that mediates depolarizationof cardiac action potentials Combines with minK to form delayed rectifier K+ channel that mediate depolarizationof cardiac action potentials Subunitscoassembleto form ATF-sensitive inward rectifier K+ channel in kidney that regulates Na–K– 2C1cotransporter activity Combines with GIRK1 to form inward rectifier K+ (Kir) channel; maintain negative membrane potential and modulate release of neurotransmitters
Missense (N470D, A561V, 1593R,G628S, V822M), intragenic deletion of nine amino acids, one frameshift, one splice donor error Twelve missense mutations, one in-frame deletion
(predispositionto ventricular arrhythmia)
KVLQTI
Bartters syndrome (renal salt
ROMK
wasting and hypokalemic acidosis) Weaver (ataxia in mice)
Mutations
KCNA1
myokymia) Long QT syndrome
Normal function
GIRK2
decreased current magnitude in oocytes injected with a 10:1 ratio of wild-type to mutant cRNA, consistent with expectations if only a single mutant subunit within a heterotetrameric channel were required to ablate normal channel function. Injection of oocytes with cRNA encoding the V408A or F184C mutant subunits induced currents with properties different than wild-type Kvl.1. The rate of deactivation and C-type inactivation was faster in homomeric V408A channels. Homomeric F184C channels had normal kinetics, but the half-point for activation was shifted by +24 mV. Coexpression of wild-type and V408 or F184C subunits reduced current by 26% and 80%, respectively, relative to current induced by injection of an equal amount of wild-type cRNA alone (Adelman et al., 1995). These findings suggest that mutations in Kvl. 1 K+ channels cause a decrease in the magnitudeof current in neurons,an effect that would likely slow the rate of action potential depolarizationand increase membrane excitability. The normal function of Kvl.1, the mutations in KCNA1 associated with EA-1 and the resulting channel dysfunctions are summarized in Table 1. A decreased function of Kvl.1 channels in the cerebellum may be the cause of ataxia, whereas a decrease in channel function in peripheral neurons is likely the cause of myokemia. Because EA-1 is episodic, future studies are required to determine what other environmental factors are important in the precipitation of attacks. Oral acetazolamide, a carbonic anhydrase inhibitor,has proved an effective treatment for EA-2 and some cases of EA-1 (Lubbers et al., 1995).However, this drug was less effective with prolongedtreatment and side
Dysfunction Decrease in Kvl.1 current, increase in neuronat excitability resulting in ataxia Decrease in 1= and prolongation of action potentials; increased risk of ventricular arrhythmias Decrease in IK, and prolongation of action potentials; increased risk of ventricular arrhythmias
Missense (A195V, S200R, M338T); two fiameshifts, two premature stops
?-likely to cause decrease in renal K+ reabsorption
Missense (G156S)
Decrease in Kir; loss of K+ selectivity and G-protein modulation
effects were notable. The mechanism of action of acetazolarnide in EA is uncertain, but may relate to a decrease in intracellularpH in the cerebellum of affected individuals (Bain et al., 1992). Pharmacological agents that either shifted the voltage dependence of Kvl.1 channel activation to more negative potentials or increased the magnitude of the current might prevent both the ataxia and myokemia characteristic of this disease. MUTATIONSIN DELAYEDRECTIFIER K+ CHANNELS,HERG ANDKVLQT1CAUSECARDIAC ARRHYTHMIAS The long QT syndrome (LQT) is an autosomal dominant inherited disorder that predisposes affected individualsto fatal cardiac arrhythmias.The basic defect in this disease is a delay in ventricular depolarization, manifest on the surface electrocardiogram (ECG) as a prolongationof the QT interval. These patients can have intermittentventriculararrhythmias,resulting in syncope or sudden death. Many individuals, however, remain completely asymptomatic. The ventricular arrhythmia associated with LQT is a polymorphic ventricular tachycardia called torsade de pointes, characterized by a twisting of the QRS axis around the isoelectric line of the body surface ECG (Dessertenne, 1966). This arrhythmia maybe caused by spatial dispersion of ventricular depolarization and an alteration in the predominance of two ectopic foci (ElSherif et al., 1996). The probable mechanism of these ectopicfoci is a micro re-entrant electrical circuit, caused
Potassium Channelopathies
by secondary depolarizations (early after-depolarizations) during the plateau phase of myocellular action potentials. This abnormal cellular effect results from a reduction in depolarizingoutward current and subsequent reactivationof L-typeCa2+channels(Januaryand Riddle, 1989). Torsade de pointes produces contractions with a frequency too rapid for adequate filling of the ventricle between beats. The resulting decrease in cardiac output causes inadequate perfusion of the brain and loss of consciousness. This arrhythmia frequently self-terminates. However, torsade de pointes can degenerate into ventricuhu-fibrillation that seldom spontaneously converts to normal rhythm and is probablythe most common cause of sudden death. Linkage analysis has identified several chromosomal loci associated with LQT. Previousstudies(Curran et al., 1995; Wang et al., 1995, 1996) used a candidate gene approach and positional cloning to identify three genes; SCN5A, HERG and KVLQTI, that cause LQT. These genes encode ion channels that modulate membrane depolarizationof the cardiac myocyte. SCN5A encodes the cardiac sodium (IN.) channel, HERG encodes subunits that form a rapid delayed rectifier K+ (Ifi) channel, and KVLQT1 encodes subunitsthat coassemble with minK to form the slow delayed rectifer K+ (IKJ channel. A candidate gene approach was used to identify the gene associatedwith chromosome7-linkedLQT (LQT2). HERG (human ether a-go-go related gene) was mapped to human chromosome 7 (Warmke and Ganetzky, 1994) and, therefore, became a candidate gene for LQT2. HERG was discovered by screening a human hippocampal cDNA library with a degenerate oligonucleotide probe homologousto eag, a DrosophilaK+channelgene. Similar to other voltage-gatedK+channels,the predicted amino acid sequence of HERG contains six putative transmembrane regions (S1–S6) and a K+-selectivepore sequence. Single strand conformation polymorphism (SSCP) analysis of DNA from affected individuals revealed several abnormal conformers and sequence analysisrevealed many mutations,includingone de now mutation (Curran et al., 1995).Northern analysisshowed that HERG was highly expressed in the heart, consistent with its role in LQT. The function of HERG was studied by heterologous expression in Xenopus oocytes. Injection of oocytes with HERG cRNA induced a K+ current that was biophysically and pharmacologicallysimilar to a delayed rectifier K+ current, 1=, previously described in isolated cardiac myocytes (Shibasaki, 1987; Sanguinetti and Jurkiewicz, 1990). A rapid onset of channel inactivationreduces the amplitude of outward channel current and is the cause of the prominent inward rectificationof the current-voltage relationship for Im (Shibasaki, 1987; Smith et al., 1996; Spector et al., 1996). Similar to other voltage-gated K+ channels, I= channels are likely to form by coassembly of four
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subunits. Therefore, it was hypothesized that mutant HERG subunits might coassemble with wild-type (WT) subunitsto form heterotetramerswith altered (or loss of,) function, a dominant-negative effect (Curran et al., 1995). This hypothesis was tested by coexpressing LQT-associatedmutant HERG subunitswith WT HERG subunits in Xenopus oocytes (Sanguinetti et al., 1996a). Deletion of base pair 1261(A1261) results in a truncated HERG protein consisting of the amino terminus and about two thirds of S1 transmembrane region. Another deletion mutant (AZ50&F508) lacked nine amino acids in the S3 transmembraneregion. No current was detected when cRNA encodingeither of these mutantproteinswas injected in oocytes. Coinjection of A1261 or AZ500F’508 cRNA with WT cRNA indicated that mutant proteins did not coassemble with WT HERG subunits. Therefore, the intragenic deletions of HERG associated with LQT result in loss of function (nullalleles),but not a dominant-negativeeffect. Missense mutations in HERG have a dominantnegative effect on 1= channel function (Sanguinetti et al., 1996a).In one mutant, a valine was substitutedfor an alanine in the S5 transmembrane region (A561V). Expression of the A561V mutant alone produced no detectable HERG current. Coexpression of WT and A561V HERG subunitsresulted in currentsthat were less than half the size of currents in oocytes expressing WT HERG alone, indicating a dominant negative effect. In addition,current induced in coinjected oocytes activated at a more negativepotentialthan normal. Another HERG missense mutation, G628S, altered the highly conserved K+-selectivepore sequence. This mutant protein did not form functional channels by itself, but coexpression of WT and G628S HERG subunitscaused almost complete suppression of current, indicating that coassembly of even a single G628S subunit in the tetrameric channel results in loss of function. Only one HERG missense mutation associated with LQT2, N470D, resulted in subunitsthat were capable of coassembly of functional channels. Injection of oocytes with N470D HERG cRNA induced currents in oocytes that were smaller and deactivated more slowly than oocytes injected with WTHERG cRNA. In addition, the voltage dependence of activation was shifted by –18 mV relative to control currents. Coexpression of cRNAs encoding N470D and WT HERG subunits resulted in a modest dominant negative effect (Sanguinetti et al., 1996a). It is likely that many other mutationsin HERG will be found that are associated with LQT2. DNA sequencing has revealed two other rnissensemutationsin HERG; one in the pore region (Benson et al., 1996)and anotherin the cyclic nucleotidebindingregion of the carboxylterminus (Satler et al., 1996) (Table 1). The common findingthat some individuals with LQT may have few, if any, syncopal episodes indicates that a reduction in HERG channel current alone is insufficient to cause torsade de pointes. Additionalenvironmentalor
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genetic factors undoubtedly contribute to the pathology of this disorder. Likely environmental factors include bradycardia, hypokalemia and certain medications (e.g. terfenidineand class III antiarrhythmicagents)that block Ifi. An activator of Ifi channelsmight amelioratethe basic molecular defect of this disorder and presumably decrease the risk of arrhythmia. Unfortunately,although specific blockers of Im channels have been developed (Sanguinetti and Jurkiewicz, 1990; Gwilt et al., 1991; Spinelli et al., 1993; Lynch et al., 1994), 1= channel activators have not been reported. The findingthat IWand HERG expressedin oocytesis paradoxically increased by an elevation of external [K+] has clinical implications. Elevation of serum [K+]using K+ supplements and spironolactone in individuals with LQT2 demonstrated a significant reduction of the QT interval (Compton et al., 1996). In addition, the dispersion of ventricular refractorinesswas reduced and abnormal T-wave morphology was corrected. Because it is difficultto maintain an elevated level of serum K+,it is unlikely that K+ supplementation will be useful for chronic treatment of LQT2. However, these findings emphasize the need to avoid hypokalemiain individuals with LQT. The most common form of inherited LQT is linked to chromosome 1lp15.5 (LQT1). A positional cloning approach was used to identify a novel gene, KVLQT1, associated with this form of LQT (Wang et al., 1996). Northern analysis indicated that KVLQT1 was highly expressed in the pancreas and heart. The predicted amino acid sequenceof KVLQT1 suggestedthat it encoded a K+ channel. Similar to other voltage-gated K+ channels, KVLQTI encodes a protein with six putative transmembrane regions and a K+-selectivepore sequence. Heterologous expression of KVLQTI in Xenopus oocytes induced a delayed rectifier K+ current with biophysical properties distinct from any known cardiac K+ current, suggesting that the protein might coassemble with another protein to form a channel with functional properties consistent with a known cardiac K+ current. A likely candidate was rninK, a small protein believed to form part of the cardiac IK, channel (Takumi et al., 1988, 1991). Coexpression of KVLQT1 with human rninK, induced a current nearly identical to IK, (Sanguinetti et al., 1996b).Similar findingswere reported for the mouse homologs of KvLQT1 and minK (Barhanin et al., 1996). It is likely that the many missense mutations in KVLQT1 associated with LQT1 (Wang et al., 1996; Russell et al., 1996) will decrease IK, by a dominantnegative effect. Thus, an activator of IK~ would be expected to reverse the primary defect of chromosome n-linked LQT. Similar to the situation described above for HERG, while blockers of 1~, channels are known (Busch et al., 1996),no specificactivatorsof this channel have been described.
MUTATIONSIN AN ATP-SENSITIVEK+CHANNEL, ROMK CAUSEDYSFUNCTIONIN RENALSALT REABSORPTION Bartter’s syndrome is an autosomal recessive renal disorder that is characterized by salt wasting, hypokalemic alkalosis, hypercalciuria and low blood pressure. Mutationsin NKCC2, the gene encoding the kidney Na– K–2C1cotransporter, were associated with this disorder in several kindreds (Simon et al., 1996a). This disorder can also be caused by mutationsin an ATP-activated K+ channel, ROMK (Simon et al., 1996b). ROMK is expressed in the thick, ascending limb of the loop of Hen16where it regulates K+ recycling and, in the distal nephron, where it mediates K+ secretion. Some ROMK mutations result in premature termination of the protein and, therefore, are a likely cause of complete loss of channel function. The functional consequences of three missense mutations in ROMK (A195V, S200R and M338T) has not been determined, but are also likely to alter, or cause loss of, channel function. Alternative splicingof ROMK results in at least three isoforms of the channel protein (Shuck et al., 1994). Because the mutations described in Bartter’s syndrome occur in a nonvariable region of the final protein, all isoforms of ROMK would be affected. It is hypothesizedthat ROMK mutations result in an inability to reabsorb K+ from the thick ascending loop to the renal tubule, causing an inhibition of Na–K–2Cl cotransport and salt wasting. Loss of salts would increase aldosteronelevels, resulting in increased Na+ absorption in exchange for K+ and H+ and hypokalemic alkalosis characteristic of this disease (Simon et al., 1996b). MUTATIONSIN A NEURALINWARDRECTIFIER K+CHANNEL,GIRK2 CAUSEDEGENERATIONOF CEREBELLA GRANULECELLSANDATAXIAIN MICE The weaver phenotype in mice is inherited as an autosomalrecessive disorder and results in severe ataxia within two weeks after birth. The weaver mutation causes a defect in neuronaldifferentiationthat is being intensely investigated. It is already known that precursor granule cells in the external germinal layer of the cerebella cortex fail to extend neurites or migrate along glial fibers to form bipolar processes, instead, they degenerate soon after birth (Rakic and Sidman, 1973).This abnormal cell death results in a greatly reduced size of the adult cerebellum.In addition,the wv/wvgenotypecauses death of dopaminergic cells in the substantial nigra, male sterility and sporadic tonic-clonic seizures (Eisenberg and Messer, 1989; Harrison and Roffler-Tarlov, 1994). Heterozygousmice are not ataxic, but have seizuresand a significantreduction in the number of granule cells. Weaver was mapped to chromosome 16 of the mouse (Reeves et al., 1989).Patil et al. (1995) used a combined physical and transcript map of the homologous segment on human chromosome 21 to isolate candidate genes for
Potassium Clmnnelopathies
this mouse disorder. DNA sequence analyses identified two potential disease genes in this region: mmb, a protein kinase and KCNJ7, which encodes an inwardlyrectifying K+ channel, GIRK2. Sequence analyses revealed no mutationsin mmb, but a singlebasepair changein GZRK2 (a G-A transition at nucleotide 953). This missense mutation results in a single amino acid substitution (G156S) in the highly conserved pore-forming (H5) region of the channel protein. GIRK2 is highly expressed in the granule cell layer of the cerebellum, the substantial nigra and the cortex (Kobayashi et al., 1995),consistent with its role in the weaver phenotype. It was proposed that a failure to maintain a hyperpolarized membrane potential by mutant GIRK2 channels impairs normal granule cell differentiation in the cerebellum and that excitotoxicitymight explain the cell loss in the substantial nigra (Patil et al., 1995). In the brain, GIRK2 subunits are believed to coassemble with GIRK1 subunits to form K+-selective, G protein-gated inward rectifier channels (Duprat et al., 1995; Kofuji et al., 1996). Both GIRK subunits are expressedin the mouse cerebellumfrom 3 days afterbirth until adulthoodand co-localizein granulecells (Slesinger et al., 1996).Physical association of GIRK1 and GIRK2 was demonstratedby co-imnmnoprecipitationof epitopetagged subunits expressed in COS-M7 cells (Navarro et al., 1996). More recently, co-irnmunoprecipitation of GIRK2 and GIRK1 was demonstrated in tissue where both proteins are expressed in the mouse brain (Liao et al., 1996). The physiological consequences of the G156S mutation in GIRK2 (wvGIRK2)were first studied in Xenopus oocytes and chinese hamster ovary (CHO) cells. The equivalent missense mutation in some voltage-gated K+ channels was previously shown to cause loss of K+ selectivity (Heginbotharn et al., 1994), raising the possibility that channels containing WVGIRK2subunts might form nonselective cation channels. As predicted, expression of homomultimeric WVGIRK2channels in oocytes and CHO cells induced a monvalent cation nonselectivecurrent that had a permeabilityratio for Na+ compared to K+ (PN,/PK)of 0.78 in oocytes (Slesingeret al., 1996) and 0.94 in CHO cells (Navarro et al., 1996). Heteromultimeric GIRK1-wvGIRK2channels expressed in CHO cells had a PN,/PKof 0.74; this compares to a PN,/PK of 0.03 for wild-type GIRK1–GIRK2 heteromulitmeric channels (Navarro et al., 1996). Therefore, under physiological conditions, WVGIRK2 channels would conduct an inward Na+ current and depolarize the resting potential, the probable cause of decreased survival of oocytes and CHO cells expressinghigh levels of this channel subunit. Channels containing WVGIRK2subunits exhibited weaker inward rectification than channels formed from WT GIRK2 subunits. In oocytes expressing GIRK1 and WVGIRK2,currents were reduced compared to cells expressing only WVGIRK2, indicating a dominant negative effect (Navarro et al., 1996; Slesinger et al.,
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1996). In addition, wvGIRK2-containingchannels were not affected by G-protein activation. Whereas the magnitudes of homomultimeric GIRK2 or heteromultimeric GIRK1-GIRK2 channels were greatly enhanced by carbachol (Slesinger et al., 1996), acetylcholine or purifiedGbg(Kofnji et al., 1996; Navarro et al., 1996), these agents had no effect on GIRK1–wvGIRK2 or homomeric WVGIRK2channels. The heterologous expression studies described above predict that GIRK1–wvGIRK2channels in granule cells of weaver mice would conduct Na+, be unresponsive to G-protein activation and have a decreased conductance compared to WT channels in normal mice. Two recent studies have examined this hypothesis in granule cells isolated from weaver mice (Kofuji et al., 1996;Surmeier et al., 1996). Kofuji et al. (1996) reported that GIRK currents in WT granule cells isolated 5–7 days after birth were activated by a nonhydrolyzable analog of GTP, whereas granule cells isolated from weaver mice were unresponsive to this agent. Weaver cells did not have a detectableGIRK current, but had a lower input resistance attributable to a Na+ leak conductance. This leak was consistent with the nonselective conductance of WVGIRK2channels characterized in oocytes. Consistent with this finding was the previous observation that the resting membrane potential of cultured weaver granule cells was depolarized (–38 mV) relative to wild-type granule cells (–61 mV) (Murtomaki et al., 1995). Surmeier et al. (1996) also reported that weaver granule cells had a pronounced reduction in GIRK current, but they could not detect a basally active, nonselectivecation current in these cells. The nonselective cation current in oocytes expressing WVGIRK2was blocked by the cation channel blockers MK-801 and QX-314 (Kofuji et al., 1996).Therefore, the ability of these drugs to alter survival and differentiation in weaver granule cells was examined. These drugs enhanced cell viability and neurite outgrowth of cultured weaver granule cells, but had no effect on cultured WT granule cells (Kofuji et al., 1996). Moreover, weaver granule cells treated with these drugs expressed TAG1, an axonal glycoprotein associated with late stages ~f granule cell differentiation that is otherwise not expressed by weaver cells. More specific blockers of Na+ channels (tetrodotoxin) and NMDA receptors (aminophosphonovalerate) were not capable of rescuing the weaver granule cells. These pharmacological experiments suggest that the altered function (i.e. nonselective cation conductance) of channels containing WVGIRK2 subunits causes premature death of granule precurser cells, not the absence of WT GIRK2 channels. However, based on the absence of a measurablenonselectivecation current in weaver granule cells, Surmeier et al. (1996) concluded that the primary defect is a failure of agonists to activate channels containingWVGIRK2subunits.They pro osed that sustained depolarization and excessive Ca1+ entry subsequentto activation of NMDA glutamate receptors leads to cell death. In normal granule cells, co-
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activation of metabotropic glutamate receptors would activate GIRK channels, resulting in a hyperpolarizing current that would counteract the glutamate receptoractivated depolarizing current. The finding that the weaver phenotype results from a single amino acid mutation in an inwardly rectifying K+ channel subunit represents a major breakthrough in the understandingof this neuronal disorder. However, many questions regarding the pathophysiologyof this disorder remain unresolved. For example, it is unclear how the presence of WT granule cells can restore normal growth and neurite extension of cultured weaver granule cells (Gao and Hatten, 1993). The elucidation of the primary defect in weaver mice may provide insights into human neural degenerative diseases (Patil et al., 1995; Surmeier et al., 1996). Degeneration of dopaminergicneurons in the substantial nigra causes Parkinson’sdisease. The same populationof neurons is affected in weaver mice. It is conceivablethat abnormal cell death in Parkinson’s disease results from dysfunction,or loss of function of a K+channelimportant in maintaining a hyperpolarized resting membrane potential. SUMMARY The firstdiscoverythat mutationsin a K+channel gene can cause an inherited disease was reported in 1994.The great diversityof K+channel genes and the importanceof these channels in modulatingcell membrane excitability implies that other inherited disorders of K+ channel function remain to be discovered. Most likely are other paroxysmal disorders of nervous and striated muscle tissue such as epilepsy, paramyotonia and cardiac arrhythmia, other than long QT syndrome. However, it is also possible that some disorders of vascular and gastric smooth muscle and nonexcitable tissues will be associated with mutations in K+ channel genes. For example, ATP-sensitive K+ (KAm) channels are not detectable in pancreatic ~-cells of patients with familiaJ persistenthypennsulinemia and hypoglycemiaof infancy (Dunne et al., 1995). This disease was mapped to the same locus as the genes encodingtwo subunits(SUR and Kir6.2) believed to coassemble to form KATPchannels (Dukes and Phillipson, 1996). An implied goal of understanding the pathophysiological basis of these disorders is to devise specific therapies for treatment or prevention. However, because many of these disorders are very rare, it is uncertain whether recent genetic and mechanistic findings will catalyse the discovery and development of specific pharmacologicaltherapies. REFERENCES AdelmanJ. P., Bond C. T., Pessia M. and Maylie J. (1995) Episodic ataxia results from voltage-dependent potassium channels with altered functions. Neuron 15: 1449–1454.
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(1996b) Murtomaki S., Trenkner E., Wright J. M., Saksela O. and Genetic heterogeneity of Bartter’s syndrome revealed by Liesi P. (1995) Increased Proteolytic Activity of the mutations in the K+ channel, ROMK. Nature Genetics 14: Granule Neurons May Contribute to Neuronal Death in the 152–156. Weaver Mouse Cerebellum. Developmental Biology 168: Slesinger P. A., Patil N., Liao Y. J., Jan Y. N., Jan L. Y. and 635-648. Cox D. R. (1996) Functional effects of the mouse weaver Navarro B., Kennedy M. E., Velimirovic B., Bhat D., Peterson A. S. and Clapham D. E. (1996) Nonselectiveand GDY- mutation on G protein-gated inwardly rectifying K+ channels. Neuron 16: 321–331. insensitiveweaver K+channels. Science 272: 1950-1953. Ophoff R. A., Terwindt G. M., Vergouwe M. N., Van Eijk R., Smith P. L., Baukrowitz T. and Yellen G. (1996) The inward rectification mechanism of the HERG cardiac potassium Oefner P. J., Hoffman S. M. G., Larnerdin J. E., channel. Nature 379: 833–836. Mohrenweiser H. W., Bulman D. E., Ferrari M., Haan J., Lindhout D., Van OmrnenG.-J. B., HofkerM. H., Ferrari M. Spector P. S., Curran M. E., Zou A., Keating M. T. and Sanguinetti M. C. (1996) Fast inactivation causes rectificaD. and Frants R. R. (1996)Familial hemiplegicmigraine and tion of the 1= channel. Journal of General Physiology 107: episodic ataxia type-2 are caused by mutations in the Ca2+ 611-619. channel gene CACNL1A4. Cell 87: 543–552.
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Pergamon PII: S0028-3908(97)00029-4
Review Potassium Channelopathies M. C. SANGUINETTI* and P. S. SPECTOR Department of Medicine, Division of Cardiologyand Eccles Program in Human Molecular Biology and Genetics, Universityof Utah, Salt Lake City, UT84112, U.S.A. (Accepted 31 December 1996)
summary-l%e molecular diversity of K+-selective channels far exceeds any other group of voltage- or ligand-gatedchannels,reflectingtheir early ancestralorigin.This diversityis mirroredby the broad spectrumof physiological functions subserved by these proteins. Potassium channels modulate the resting potential and action potential duration of neurons, myocytes and endocrine cells and stabilize the membrane potential of excitable and nonexcitablecells. In additionto channeldiversity,differentialcellular expressionof K+channels determinesthe specific electrical responsesto stimuli in a particular cell or tissue. This studyreviews the recent genetic and physiological studies of congenital disorderscaused by mutations in genes encoding K+channels. These include the human disorders of episodic ataxia with rnyokymia, long QT syndrome and Bartter’s syndrome,and weaver ataxia in mice. An understandingof the molecularbasis of these diseasescould facilitate the discovery and development of specific pharmacologicaltherapies. 01997 Elsevier Science Ltd. Keywords—Episodic ataxia, long QT syndrome,Bartters syndrome,weaver.
MUTATIONSIN A NEURALDELAYEDRECTIFIERK+ Litt et al. (1994) used linkage studies to localize the CHANNEL,KV1.1CAUSEEPISODICATAXIAAND EA-1 gene to chromosome 12p, where three related K+ MYOKYMIA channelgenes (KCNAl, KCNA5, KCNA6) had previously Episodic ataxia (EA) is a rare autosomal dominant been mapped. Mutational analysis of KCNA1 in seven necrologic disorder characterized by intermittent attacks families identified six different missense mutations of generalized ataxia, caused by physical or emotional (V174F, F184C, R239S, F2491,E325D, V408A) in this stress. Two types of EA are recognized based on the gene (Browne et al., 1994, 1995). One additional family length and severity of the attacks. Ataxia lasts for only a with EA-1 was shown to result from another missense few seconds or minutes in EA-1 and is associated with mutation (C677T) in KCNA1 (Comu et al., 1996). continuous motor neuron activity, which causes muscle KCNA1 encodes the Kvl.1 delayed rectifier K+ channel rippling (myokymia) between and during attacks (Van that is expressed in the cerebellum and peripheral Dyke et al., 1975). Ataxia lasts for several hours to nervous system of the rat (Beckh and Pongs, 1990). several days in EA-2 and is associated with nystagmus Because affected members in all families had similar and cerebella atrophy.Linkage analysismapped the EA- symptoms, it was not possible to make phenotype– genotypecorrelations.Browne et al. (1995)hypothesized 2 disease gene to chromosome 19p13, the same region that mutationsin KCNAI would likely decrease function associated with familial hemiplegic migraine (FHM), of the Kvl.1 channels by a dominant negative effect. suggestingthe possibility that EA-2 and FHM are allelic Adelman et al. (1995) studied the physiological disorders.This was confirmedrecently with the discovery consequences of EA-1 associated mutations in KCNAI that both disorders are caused by mutations in CACN- by expressingthe mutant Kvl.1 proteins, either alone or LIA4, a brain-specificP/Q-type Ca2+channel (Ophoff et in combination with wild-type subunits in Xenopus al., 1996). oocytes. Four of the mutant Kvl.1 subunits (V174F, . R239S, F2491, E325D) did not induce expression of *To+whomcorrespondenceshouldbe addressed.Tel: 801-585- functional channels, but suppressed current when coex6336; Fax: 801-585-3501;e-mail: mike.sanguinetti@gene- pressed with wild-type subunits; a dominant negative effect. The R239S and E325D mutant subunits greatly tics.utah.edu 755
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M. C. Sanguinetti and P. S. Spector Table 1. Summary of diseases caused by mutations in K+ channel genes
Disease EA-1 (episodic ataxia and
Mutated K+ channel gene
Coassembles as tetrarner to form neural delayed rectifier K+channel
Missense (V174F,F184C, R239S, F2491,E325D, V408A, C677T)
HERG
Coassembles as tetramer to form rapid delayed rectifier K+ channel that mediates depolarizationof cardiac action potentials Combines with minK to form delayed rectifier K+ channel that mediate depolarizationof cardiac action potentials Subunitscoassembleto form ATF-sensitive inward rectifier K+ channel in kidney that regulates Na–K– 2C1cotransporter activity Combines with GIRK1 to form inward rectifier K+ (Kir) channel; maintain negative membrane potential and modulate release of neurotransmitters
Missense (N470D, A561V, 1593R,G628S, V822M), intragenic deletion of nine amino acids, one frameshift, one splice donor error Twelve missense mutations, one in-frame deletion
(predispositionto ventricular arrhythmia)
KVLQTI
Bartters syndrome (renal salt
ROMK
wasting and hypokalemic acidosis) Weaver (ataxia in mice)
Mutations
KCNA1
myokymia) Long QT syndrome
Normal function
GIRK2
decreased current magnitude in oocytes injected with a 10:1 ratio of wild-type to mutant cRNA, consistent with expectations if only a single mutant subunit within a heterotetrameric channel were required to ablate normal channel function. Injection of oocytes with cRNA encoding the V408A or F184C mutant subunits induced currents with properties different than wild-type Kvl.1. The rate of deactivation and C-type inactivation was faster in homomeric V408A channels. Homomeric F184C channels had normal kinetics, but the half-point for activation was shifted by +24 mV. Coexpression of wild-type and V408 or F184C subunits reduced current by 26% and 80%, respectively, relative to current induced by injection of an equal amount of wild-type cRNA alone (Adelman et al., 1995). These findings suggest that mutations in Kvl. 1 K+ channels cause a decrease in the magnitudeof current in neurons,an effect that would likely slow the rate of action potential depolarizationand increase membrane excitability. The normal function of Kvl.1, the mutations in KCNA1 associated with EA-1 and the resulting channel dysfunctions are summarized in Table 1. A decreased function of Kvl.1 channels in the cerebellum may be the cause of ataxia, whereas a decrease in channel function in peripheral neurons is likely the cause of myokemia. Because EA-1 is episodic, future studies are required to determine what other environmental factors are important in the precipitation of attacks. Oral acetazolamide, a carbonic anhydrase inhibitor,has proved an effective treatment for EA-2 and some cases of EA-1 (Lubbers et al., 1995).However, this drug was less effective with prolongedtreatment and side
Dysfunction Decrease in Kvl.1 current, increase in neuronat excitability resulting in ataxia Decrease in 1= and prolongation of action potentials; increased risk of ventricular arrhythmias Decrease in IK, and prolongation of action potentials; increased risk of ventricular arrhythmias
Missense (A195V, S200R, M338T); two fiameshifts, two premature stops
?-likely to cause decrease in renal K+ reabsorption
Missense (G156S)
Decrease in Kir; loss of K+ selectivity and G-protein modulation
effects were notable. The mechanism of action of acetazolarnide in EA is uncertain, but may relate to a decrease in intracellularpH in the cerebellum of affected individuals (Bain et al., 1992). Pharmacological agents that either shifted the voltage dependence of Kvl.1 channel activation to more negative potentials or increased the magnitude of the current might prevent both the ataxia and myokemia characteristic of this disease. MUTATIONSIN DELAYEDRECTIFIER K+ CHANNELS,HERG ANDKVLQT1CAUSECARDIAC ARRHYTHMIAS The long QT syndrome (LQT) is an autosomal dominant inherited disorder that predisposes affected individualsto fatal cardiac arrhythmias.The basic defect in this disease is a delay in ventricular depolarization, manifest on the surface electrocardiogram (ECG) as a prolongationof the QT interval. These patients can have intermittentventriculararrhythmias,resulting in syncope or sudden death. Many individuals, however, remain completely asymptomatic. The ventricular arrhythmia associated with LQT is a polymorphic ventricular tachycardia called torsade de pointes, characterized by a twisting of the QRS axis around the isoelectric line of the body surface ECG (Dessertenne, 1966). This arrhythmia maybe caused by spatial dispersion of ventricular depolarization and an alteration in the predominance of two ectopic foci (ElSherif et al., 1996). The probable mechanism of these ectopicfoci is a micro re-entrant electrical circuit, caused
Potassium Channelopathies
by secondary depolarizations (early after-depolarizations) during the plateau phase of myocellular action potentials. This abnormal cellular effect results from a reduction in depolarizingoutward current and subsequent reactivationof L-typeCa2+channels(Januaryand Riddle, 1989). Torsade de pointes produces contractions with a frequency too rapid for adequate filling of the ventricle between beats. The resulting decrease in cardiac output causes inadequate perfusion of the brain and loss of consciousness. This arrhythmia frequently self-terminates. However, torsade de pointes can degenerate into ventricuhu-fibrillation that seldom spontaneously converts to normal rhythm and is probablythe most common cause of sudden death. Linkage analysis has identified several chromosomal loci associated with LQT. Previousstudies(Curran et al., 1995; Wang et al., 1995, 1996) used a candidate gene approach and positional cloning to identify three genes; SCN5A, HERG and KVLQTI, that cause LQT. These genes encode ion channels that modulate membrane depolarizationof the cardiac myocyte. SCN5A encodes the cardiac sodium (IN.) channel, HERG encodes subunits that form a rapid delayed rectifier K+ (Ifi) channel, and KVLQT1 encodes subunitsthat coassemble with minK to form the slow delayed rectifer K+ (IKJ channel. A candidate gene approach was used to identify the gene associatedwith chromosome7-linkedLQT (LQT2). HERG (human ether a-go-go related gene) was mapped to human chromosome 7 (Warmke and Ganetzky, 1994) and, therefore, became a candidate gene for LQT2. HERG was discovered by screening a human hippocampal cDNA library with a degenerate oligonucleotide probe homologousto eag, a DrosophilaK+channelgene. Similar to other voltage-gatedK+channels,the predicted amino acid sequence of HERG contains six putative transmembrane regions (S1–S6) and a K+-selectivepore sequence. Single strand conformation polymorphism (SSCP) analysis of DNA from affected individuals revealed several abnormal conformers and sequence analysisrevealed many mutations,includingone de now mutation (Curran et al., 1995).Northern analysisshowed that HERG was highly expressed in the heart, consistent with its role in LQT. The function of HERG was studied by heterologous expression in Xenopus oocytes. Injection of oocytes with HERG cRNA induced a K+ current that was biophysically and pharmacologicallysimilar to a delayed rectifier K+ current, 1=, previously described in isolated cardiac myocytes (Shibasaki, 1987; Sanguinetti and Jurkiewicz, 1990). A rapid onset of channel inactivationreduces the amplitude of outward channel current and is the cause of the prominent inward rectificationof the current-voltage relationship for Im (Shibasaki, 1987; Smith et al., 1996; Spector et al., 1996). Similar to other voltage-gated K+ channels, I= channels are likely to form by coassembly of four
757
subunits. Therefore, it was hypothesized that mutant HERG subunits might coassemble with wild-type (WT) subunitsto form heterotetramerswith altered (or loss of,) function, a dominant-negative effect (Curran et al., 1995). This hypothesis was tested by coexpressing LQT-associatedmutant HERG subunitswith WT HERG subunits in Xenopus oocytes (Sanguinetti et al., 1996a). Deletion of base pair 1261(A1261) results in a truncated HERG protein consisting of the amino terminus and about two thirds of S1 transmembrane region. Another deletion mutant (AZ50&F508) lacked nine amino acids in the S3 transmembraneregion. No current was detected when cRNA encodingeither of these mutantproteinswas injected in oocytes. Coinjection of A1261 or AZ500F’508 cRNA with WT cRNA indicated that mutant proteins did not coassemble with WT HERG subunits. Therefore, the intragenic deletions of HERG associated with LQT result in loss of function (nullalleles),but not a dominant-negativeeffect. Missense mutations in HERG have a dominantnegative effect on 1= channel function (Sanguinetti et al., 1996a).In one mutant, a valine was substitutedfor an alanine in the S5 transmembrane region (A561V). Expression of the A561V mutant alone produced no detectable HERG current. Coexpression of WT and A561V HERG subunitsresulted in currentsthat were less than half the size of currents in oocytes expressing WT HERG alone, indicating a dominant negative effect. In addition,current induced in coinjected oocytes activated at a more negativepotentialthan normal. Another HERG missense mutation, G628S, altered the highly conserved K+-selectivepore sequence. This mutant protein did not form functional channels by itself, but coexpression of WT and G628S HERG subunitscaused almost complete suppression of current, indicating that coassembly of even a single G628S subunit in the tetrameric channel results in loss of function. Only one HERG missense mutation associated with LQT2, N470D, resulted in subunitsthat were capable of coassembly of functional channels. Injection of oocytes with N470D HERG cRNA induced currents in oocytes that were smaller and deactivated more slowly than oocytes injected with WTHERG cRNA. In addition, the voltage dependence of activation was shifted by –18 mV relative to control currents. Coexpression of cRNAs encoding N470D and WT HERG subunits resulted in a modest dominant negative effect (Sanguinetti et al., 1996a). It is likely that many other mutationsin HERG will be found that are associated with LQT2. DNA sequencing has revealed two other rnissensemutationsin HERG; one in the pore region (Benson et al., 1996)and anotherin the cyclic nucleotidebindingregion of the carboxylterminus (Satler et al., 1996) (Table 1). The common findingthat some individuals with LQT may have few, if any, syncopal episodes indicates that a reduction in HERG channel current alone is insufficient to cause torsade de pointes. Additionalenvironmentalor
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M. C. Sanguinetti and P. S. Spector
genetic factors undoubtedly contribute to the pathology of this disorder. Likely environmental factors include bradycardia, hypokalemia and certain medications (e.g. terfenidineand class III antiarrhythmicagents)that block Ifi. An activator of Ifi channelsmight amelioratethe basic molecular defect of this disorder and presumably decrease the risk of arrhythmia. Unfortunately,although specific blockers of Im channels have been developed (Sanguinetti and Jurkiewicz, 1990; Gwilt et al., 1991; Spinelli et al., 1993; Lynch et al., 1994), 1= channel activators have not been reported. The findingthat IWand HERG expressedin oocytesis paradoxically increased by an elevation of external [K+] has clinical implications. Elevation of serum [K+]using K+ supplements and spironolactone in individuals with LQT2 demonstrated a significant reduction of the QT interval (Compton et al., 1996). In addition, the dispersion of ventricular refractorinesswas reduced and abnormal T-wave morphology was corrected. Because it is difficultto maintain an elevated level of serum K+,it is unlikely that K+ supplementation will be useful for chronic treatment of LQT2. However, these findings emphasize the need to avoid hypokalemiain individuals with LQT. The most common form of inherited LQT is linked to chromosome 1lp15.5 (LQT1). A positional cloning approach was used to identify a novel gene, KVLQT1, associated with this form of LQT (Wang et al., 1996). Northern analysis indicated that KVLQT1 was highly expressed in the pancreas and heart. The predicted amino acid sequenceof KVLQT1 suggestedthat it encoded a K+ channel. Similar to other voltage-gated K+ channels, KVLQTI encodes a protein with six putative transmembrane regions and a K+-selectivepore sequence. Heterologous expression of KVLQTI in Xenopus oocytes induced a delayed rectifier K+ current with biophysical properties distinct from any known cardiac K+ current, suggesting that the protein might coassemble with another protein to form a channel with functional properties consistent with a known cardiac K+ current. A likely candidate was rninK, a small protein believed to form part of the cardiac IK, channel (Takumi et al., 1988, 1991). Coexpression of KVLQT1 with human rninK, induced a current nearly identical to IK, (Sanguinetti et al., 1996b).Similar findingswere reported for the mouse homologs of KvLQT1 and minK (Barhanin et al., 1996). It is likely that the many missense mutations in KVLQT1 associated with LQT1 (Wang et al., 1996; Russell et al., 1996) will decrease IK, by a dominantnegative effect. Thus, an activator of IK~ would be expected to reverse the primary defect of chromosome n-linked LQT. Similar to the situation described above for HERG, while blockers of 1~, channels are known (Busch et al., 1996),no specificactivatorsof this channel have been described.
MUTATIONSIN AN ATP-SENSITIVEK+CHANNEL, ROMK CAUSEDYSFUNCTIONIN RENALSALT REABSORPTION Bartter’s syndrome is an autosomal recessive renal disorder that is characterized by salt wasting, hypokalemic alkalosis, hypercalciuria and low blood pressure. Mutationsin NKCC2, the gene encoding the kidney Na– K–2C1cotransporter, were associated with this disorder in several kindreds (Simon et al., 1996a). This disorder can also be caused by mutationsin an ATP-activated K+ channel, ROMK (Simon et al., 1996b). ROMK is expressed in the thick, ascending limb of the loop of Hen16where it regulates K+ recycling and, in the distal nephron, where it mediates K+ secretion. Some ROMK mutations result in premature termination of the protein and, therefore, are a likely cause of complete loss of channel function. The functional consequences of three missense mutations in ROMK (A195V, S200R and M338T) has not been determined, but are also likely to alter, or cause loss of, channel function. Alternative splicingof ROMK results in at least three isoforms of the channel protein (Shuck et al., 1994). Because the mutations described in Bartter’s syndrome occur in a nonvariable region of the final protein, all isoforms of ROMK would be affected. It is hypothesizedthat ROMK mutations result in an inability to reabsorb K+ from the thick ascending loop to the renal tubule, causing an inhibition of Na–K–2Cl cotransport and salt wasting. Loss of salts would increase aldosteronelevels, resulting in increased Na+ absorption in exchange for K+ and H+ and hypokalemic alkalosis characteristic of this disease (Simon et al., 1996b). MUTATIONSIN A NEURALINWARDRECTIFIER K+CHANNEL,GIRK2 CAUSEDEGENERATIONOF CEREBELLA GRANULECELLSANDATAXIAIN MICE The weaver phenotype in mice is inherited as an autosomalrecessive disorder and results in severe ataxia within two weeks after birth. The weaver mutation causes a defect in neuronaldifferentiationthat is being intensely investigated. It is already known that precursor granule cells in the external germinal layer of the cerebella cortex fail to extend neurites or migrate along glial fibers to form bipolar processes, instead, they degenerate soon after birth (Rakic and Sidman, 1973).This abnormal cell death results in a greatly reduced size of the adult cerebellum.In addition,the wv/wvgenotypecauses death of dopaminergic cells in the substantial nigra, male sterility and sporadic tonic-clonic seizures (Eisenberg and Messer, 1989; Harrison and Roffler-Tarlov, 1994). Heterozygousmice are not ataxic, but have seizuresand a significantreduction in the number of granule cells. Weaver was mapped to chromosome 16 of the mouse (Reeves et al., 1989).Patil et al. (1995) used a combined physical and transcript map of the homologous segment on human chromosome 21 to isolate candidate genes for
Potassium Clmnnelopathies
this mouse disorder. DNA sequence analyses identified two potential disease genes in this region: mmb, a protein kinase and KCNJ7, which encodes an inwardlyrectifying K+ channel, GIRK2. Sequence analyses revealed no mutationsin mmb, but a singlebasepair changein GZRK2 (a G-A transition at nucleotide 953). This missense mutation results in a single amino acid substitution (G156S) in the highly conserved pore-forming (H5) region of the channel protein. GIRK2 is highly expressed in the granule cell layer of the cerebellum, the substantial nigra and the cortex (Kobayashi et al., 1995),consistent with its role in the weaver phenotype. It was proposed that a failure to maintain a hyperpolarized membrane potential by mutant GIRK2 channels impairs normal granule cell differentiation in the cerebellum and that excitotoxicitymight explain the cell loss in the substantial nigra (Patil et al., 1995). In the brain, GIRK2 subunits are believed to coassemble with GIRK1 subunits to form K+-selective, G protein-gated inward rectifier channels (Duprat et al., 1995; Kofuji et al., 1996). Both GIRK subunits are expressedin the mouse cerebellumfrom 3 days afterbirth until adulthoodand co-localizein granulecells (Slesinger et al., 1996).Physical association of GIRK1 and GIRK2 was demonstratedby co-imnmnoprecipitationof epitopetagged subunits expressed in COS-M7 cells (Navarro et al., 1996). More recently, co-irnmunoprecipitation of GIRK2 and GIRK1 was demonstrated in tissue where both proteins are expressed in the mouse brain (Liao et al., 1996). The physiological consequences of the G156S mutation in GIRK2 (wvGIRK2)were first studied in Xenopus oocytes and chinese hamster ovary (CHO) cells. The equivalent missense mutation in some voltage-gated K+ channels was previously shown to cause loss of K+ selectivity (Heginbotharn et al., 1994), raising the possibility that channels containing WVGIRK2subunts might form nonselective cation channels. As predicted, expression of homomultimeric WVGIRK2channels in oocytes and CHO cells induced a monvalent cation nonselectivecurrent that had a permeabilityratio for Na+ compared to K+ (PN,/PK)of 0.78 in oocytes (Slesingeret al., 1996) and 0.94 in CHO cells (Navarro et al., 1996). Heteromultimeric GIRK1-wvGIRK2channels expressed in CHO cells had a PN,/PKof 0.74; this compares to a PN,/PK of 0.03 for wild-type GIRK1–GIRK2 heteromulitmeric channels (Navarro et al., 1996). Therefore, under physiological conditions, WVGIRK2 channels would conduct an inward Na+ current and depolarize the resting potential, the probable cause of decreased survival of oocytes and CHO cells expressinghigh levels of this channel subunit. Channels containing WVGIRK2subunits exhibited weaker inward rectification than channels formed from WT GIRK2 subunits. In oocytes expressing GIRK1 and WVGIRK2,currents were reduced compared to cells expressing only WVGIRK2, indicating a dominant negative effect (Navarro et al., 1996; Slesinger et al.,
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1996). In addition, wvGIRK2-containingchannels were not affected by G-protein activation. Whereas the magnitudes of homomultimeric GIRK2 or heteromultimeric GIRK1-GIRK2 channels were greatly enhanced by carbachol (Slesinger et al., 1996), acetylcholine or purifiedGbg(Kofnji et al., 1996; Navarro et al., 1996), these agents had no effect on GIRK1–wvGIRK2 or homomeric WVGIRK2channels. The heterologous expression studies described above predict that GIRK1–wvGIRK2channels in granule cells of weaver mice would conduct Na+, be unresponsive to G-protein activation and have a decreased conductance compared to WT channels in normal mice. Two recent studies have examined this hypothesis in granule cells isolated from weaver mice (Kofuji et al., 1996;Surmeier et al., 1996). Kofuji et al. (1996) reported that GIRK currents in WT granule cells isolated 5–7 days after birth were activated by a nonhydrolyzable analog of GTP, whereas granule cells isolated from weaver mice were unresponsive to this agent. Weaver cells did not have a detectableGIRK current, but had a lower input resistance attributable to a Na+ leak conductance. This leak was consistent with the nonselective conductance of WVGIRK2channels characterized in oocytes. Consistent with this finding was the previous observation that the resting membrane potential of cultured weaver granule cells was depolarized (–38 mV) relative to wild-type granule cells (–61 mV) (Murtomaki et al., 1995). Surmeier et al. (1996) also reported that weaver granule cells had a pronounced reduction in GIRK current, but they could not detect a basally active, nonselectivecation current in these cells. The nonselective cation current in oocytes expressing WVGIRK2was blocked by the cation channel blockers MK-801 and QX-314 (Kofuji et al., 1996).Therefore, the ability of these drugs to alter survival and differentiation in weaver granule cells was examined. These drugs enhanced cell viability and neurite outgrowth of cultured weaver granule cells, but had no effect on cultured WT granule cells (Kofuji et al., 1996). Moreover, weaver granule cells treated with these drugs expressed TAG1, an axonal glycoprotein associated with late stages ~f granule cell differentiation that is otherwise not expressed by weaver cells. More specific blockers of Na+ channels (tetrodotoxin) and NMDA receptors (aminophosphonovalerate) were not capable of rescuing the weaver granule cells. These pharmacological experiments suggest that the altered function (i.e. nonselective cation conductance) of channels containing WVGIRK2 subunits causes premature death of granule precurser cells, not the absence of WT GIRK2 channels. However, based on the absence of a measurablenonselectivecation current in weaver granule cells, Surmeier et al. (1996) concluded that the primary defect is a failure of agonists to activate channels containingWVGIRK2subunits.They pro osed that sustained depolarization and excessive Ca1+ entry subsequentto activation of NMDA glutamate receptors leads to cell death. In normal granule cells, co-
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activation of metabotropic glutamate receptors would activate GIRK channels, resulting in a hyperpolarizing current that would counteract the glutamate receptoractivated depolarizing current. The finding that the weaver phenotype results from a single amino acid mutation in an inwardly rectifying K+ channel subunit represents a major breakthrough in the understandingof this neuronal disorder. However, many questions regarding the pathophysiologyof this disorder remain unresolved. For example, it is unclear how the presence of WT granule cells can restore normal growth and neurite extension of cultured weaver granule cells (Gao and Hatten, 1993). The elucidation of the primary defect in weaver mice may provide insights into human neural degenerative diseases (Patil et al., 1995; Surmeier et al., 1996). Degeneration of dopaminergicneurons in the substantial nigra causes Parkinson’sdisease. The same populationof neurons is affected in weaver mice. It is conceivablethat abnormal cell death in Parkinson’s disease results from dysfunction,or loss of function of a K+channelimportant in maintaining a hyperpolarized resting membrane potential. SUMMARY The firstdiscoverythat mutationsin a K+channel gene can cause an inherited disease was reported in 1994.The great diversityof K+channel genes and the importanceof these channels in modulatingcell membrane excitability implies that other inherited disorders of K+ channel function remain to be discovered. Most likely are other paroxysmal disorders of nervous and striated muscle tissue such as epilepsy, paramyotonia and cardiac arrhythmia, other than long QT syndrome. However, it is also possible that some disorders of vascular and gastric smooth muscle and nonexcitable tissues will be associated with mutations in K+ channel genes. For example, ATP-sensitive K+ (KAm) channels are not detectable in pancreatic ~-cells of patients with familiaJ persistenthypennsulinemia and hypoglycemiaof infancy (Dunne et al., 1995). This disease was mapped to the same locus as the genes encodingtwo subunits(SUR and Kir6.2) believed to coassemble to form KATPchannels (Dukes and Phillipson, 1996). An implied goal of understanding the pathophysiological basis of these disorders is to devise specific therapies for treatment or prevention. However, because many of these disorders are very rare, it is uncertain whether recent genetic and mechanistic findings will catalyse the discovery and development of specific pharmacologicaltherapies. REFERENCES AdelmanJ. P., Bond C. T., Pessia M. and Maylie J. (1995) Episodic ataxia results from voltage-dependent potassium channels with altered functions. Neuron 15: 1449–1454.
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