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Pathophysiology of ion channel mutations
326
Pathophysiology of ion channel mutations Mark T Keating* and Michael C Sanguinettit The past year has seen significant advances in our understanding of ion channel disorders. The highlights of these advances include a detailed delineation of the molecular mechanisms underlying inherited cardiac arrhythmias and the discovery that ion channel mutations can contribute to neural development and neurodegeneration.
Address *Howard Hughes Medical Institute, University of Utah, Building 533, Room 6110B, Salt Lake City, Utah 84112, USA
tEccles Program in Human Molecular Biology and Genetics, University of Utah, Building 533, Room 4220, Salt Lake City, Utah 84112, USA
Current Opinion in Genetics & Development 1996, 6:326-333 © Current Biology Ltd ISSN 0959-437X
Abbreviations HERG
LQT CFTR
AChR
humanether-a-go-go related gene long QT cystic fibrosis transmembrane conductance regulator acetylcholine receptor
Introduction Ion channels regulate a wide variety of cellular functiolas. Not surprisingly, it has become clear that mutations in genes encoding ion channels contribute to a wide variety of pathological disorders in both excitable and non-excitable cells. These conditions include disorders of cellular excitability in the heart (long Q T [LQT] syndrome, an inherited cardiac arrhythmia), skeletal muscle (myotonia and periodic paralysis), and the central nervous system (episodic ataxia and seizures). Mutations and genes encoding ion channels also contribute to disorders of fluid and electrolyte metabolism, including hypertension and nephrogenic diabetes insipidus. Ion channel dysfunction can also contribute to neural development and degeneration. T h e two major discoveries in the past year were the discovery of the molecular mechanisms underlying life-threatening cardiac arrhythmias and the role of ion channels in neural development and degeneration. Voltage-gated ion channel mutations and cellular excitability Loss, alteration, and dominant-negative suppression of potassium and chloride channel function can lead to delayed cellular repolarization and increased excitability Voltage-gated potassium channels comprise a large family of proteins with varying functions and distinct structural motifs. A common class of potassium channel c~ subunits are thought to contain six membrane-spanning segments, SI-S 6, and a pore-forming domain located between S 5 and $6 (Fig. 1). T h e fourth membrane-spanning segment, $4, contains a positively charged amino acid residue at every
third position of the putative cc helix and is thought to be critical for voltage sensitivity. As functional potassium channels are formed from the co-assembly of four o~ subunits, either as homotetramers or heterotetramers, some mutations may have a dominant-negative effect. L Q T syndrome is an inherited disorder that causes syncope and sudden death in young, otherwise healthy, individuals. T h e symptoms result from episodic cardiac arrhythmias, specifically torsade de pointes and vcntricular fibrillation. T h e only other phenotypic abnormality in affected individuals is subtle and variable prolongation of the Q T interval on surface electrocardiograms, an indication of abnormal cardiac repolarization. Molecular genetic studies of familial and sporadic cases have demonstrated that missense mutations and small deletions in cardiac voltage-gated potassium channel genes can lead to this disorder. A positional/candidate gene approach was used to demonstrate that mutations in the human ether-a-go-go related gene (HERG; Table 1) cause chromosome 7 linked L Q T [1,2*'], whereas a pure positional cloning approach was used to identify a novel potassium channel gene, KVLQT1, and demonstrate that mutations in this gene cause the chromosome 11 linked form of this disorder [3°°]. Heterologous expression of HERG in Xenopus oocytes led to the discovery that this gene encodes subunits which form a cardiac delayed rectifier potassium channel, IKr [4°°]. T h e biophysical and pharmacological properties of HERG homotetramers expressed in oocytes appear nearly identical to those of IKr in cardiac myocytes [5,6]. Previous studies have indicated that a pharmacological block of IKr is a common cause of drug-induced arrhythmias, providing a mechanistic link between inherited and acquired cardiac arrhythmias. Co-expression of mutant and wild-type H E R G channels has led to the discovery that most LQT-associated mutations cause dominant-negative loss of H E R G function [7°]. T h e potency of the dominant-negative effect is variable; some mutants have relatively little or no effect on normal H E R G subunits, whereas others lead to a near total disruption of t I E R G function. Thus, these mutations lead to a spectrum of H E R G sodium channel dysfunction in LQT. The function of the protein encoded by KVLQT1 is not yet known, but it is likely to be a voltage-gated potassium channel which is important for myocellular repolarization. These data are consistent with the hypothesis that I , Q T results from delayed myocellular repolarization, secondary depolarizations (afterdepolarizations), and increased risk of cardiac arrhythmia. A similar mechanism has been implicated in a disorder of the central nervous system--episodic ataxia/myokymia
Pathophysiology of ion channel mutations Keating and Sanguinetti 32?
Figure 1
(a)
Potassium $1
S2
S3
S4
S6 Extracellular
rCOOH_ (b) Chloride
(c)
4
COOH-
Sodium/Calcium DI
DII
Dill
DIV Extracellular
l NH3+
Intracellular
~ C O O H ,~"1996CurrentOpinioninGenetics&Development
Predicted topology for (z subunits of voltage-gated ion channels: (a) potassium, (b) chloride and (c) sodium/calcium channels. Most functional potassium channels are formed by the coassembly of four identical subunits, analagous to a single sodium or calcium c~subunit. The 'barrels' (e.g. S1-S 6 in the potassium channel) represent (z helices. The pores of these channels in (a) and (b) are formed by the loops between segments $5 and $6; the $4 transmembrane region is thought to be the voltage sensor. The topology of the chloride channel subunits is uncertain, but functional chloride channels are probably formed by the coassembly of 4 subunits.
328 Genetics of disease
syndrome. In this disorder, individuals suffer from episodic bouts of severe ataxia and mild myokymia (muscle rippling). Molecular genetic studies have implicated missense mutations in KCNA1, a voltage-gated potassium channel gene expressed in cerebellar neurons [8"*,9]. Physiological studies indicate that disease-associated mutations in KCNA1 lead to altered channel function (e.g. faster inactivation or altered voltage dependence of activation) and dominant-negative suppression of repolarizing K + current [10"]. Potassium channel mutations have not yet been implicated in a disorder of skeletal muscle, but a similar mechanism is thought to be involved in the pathogenesis of myotonia congenita, a disorder that causes episodic muscle spasms. Molecular genetic studies have shown that this disorder results from mutations in a skeletal muscle chloride channel gene, CLC1 [11-16]. Chloride channels are also thought to form oligomers and physiological studies indicate that these mutations cause dominant-negative suppression of chloride current [17"]. One of the more exciting aspects of these studies has been their therapeutic implications. It is likely, for example, that the risk of arrhythmia in an individual with mutations of HERG or KVLQTI can be reduced with potassium channel opening agents or even supplementation with potassium itself. H E R G is paradoxically sensitive to extracellular potassium concentration; increasing extracellular potassium in a physiological range increases outward potassium current through H E R G channels [4"*]. The precise mechanism of this phenomenon is not known, but probably involves allosteric interactions between potassium ions and the extracellular domain of the channel, resulting in an increase in open probability. Preliminary data suggest that modest increases in serum potassium concentration correct cardiac repolarization abnormalities in patients with chromosome 7 linked LQT. This therapeutic approach should also work for patients with drug-induced L Q T and may be effective for L Q T resulting from mutations in other genes (KVLQT1, SCNSA, etc.). Gain of function mutations in sodium channel genes can cause abnormal cellular repolarization and increased excitability Activation of voltage-gated sodium channels initiates the action potential in muscle and neurons. T h e ct subunit of these channels is thought to contain four major domains (DI-DIV), each of which is thought to contain six membrane-spanning segments. A single sodium channel ct subunit is sufficient for channel function, but some functional characteristics arc modulated by other subunits.
Molecular genetic studies have shown that missense mutations and small deletions of the cardiac sodium channel gene SCN5A are a rare cause of inherited L Q T [18*',19]. T h e mutations that have been identified to date have been in the interdomain segment between D i l l and DI'v:
This region is important for sodium channel inactivation. Expression of mutant sodium channels in Xenopus oocytes, however, did not show significant changes in the kinetics of channel inactivation. Instead, a subtle maintained sodium current was detected. Single channel recordings indicated infrequent late reopenings, leading to depolarizing inward current late in the action potential [20*]. Thus, LQT-associated mutations in SCN5A destabilize the channel's inactivation gate. Continued depolarizing sodium current late in the plateau phase of the myocellular action potential presumably prolongs action potential duration, increasing the risk of afterdepolarizations and arrhythmia. A similar mechanism was previously demonstrated for hyperkalemic periodic paralysis, a disorder of skeletal muscle. This disorder results from gain of function mutations in a skeletal muscle sodium channel gene leading to episodic skeletal muscle excitation (myotonia) and periodic paralysis [21-23]. Voltage-gated calcium channel ct subunits have a structure that is similar to sodium channel et subunits. Molecular genetic studies have implicated missense mutations in voltage-gated L-type calcium channel ct subunits in the skeletal muscle disorder hypokalemic periodic paralysis [24*',25"',26]. In this disorder, individuals suffer from episodic myotonia followed by paralysis in the setting of low serum potassium concentrations. Although the precise functional consequences of these mutations are not clear, it is likely that these mutations increase inward calcium current and prolong cellular repolarization. Ion c h a n n e l m u t a t i o n s a f f e c t i n g w a t e r a n d electrolyte homeostasis Epithelial sodium channels and hypertension T h e epithelial sodium channel is expressed in the kidney and colon and is critical for sodium resorption. T h e channel comprises ~, 13 and T subunits and is activated by angiotensin [27"*,28]. Molecular genetic studies have shown that mutations in epithelial sodium channel genes cause Liddle syndrome, a rare inherited disorder that causes salt-sensitive hypertension [29**,30*]. Physiological studies of mutant channel subunits indicate an increase in sodium absorption [31",32"]. Surprisingly, the mutant mechanism does not appear to involve altered biophysical properties; instead, the molecular mechanism appears to be increased numbers of epithelial sodium channel subunits, probably caused by altered protein degradation. The aquaporin-2 water channel and diabetes insipidus T h e aquaporin-2 water channel has a topology similar to potassium channels; the predicted structure suggests that the amino and carboxyl termini are located in the intracellular compartment and the protein has six putative membrane-spanning domains [33]. These channels are known to be important for vasopressin-induced resorption of water by the kidney. Molecular genetic studies have
Pathophysiology of ion channel mutations Keating and Sanguinetti 329
Table 1 Pathophysiology of ion channel mutations. Channels
Gene
Protein
Voltage-gated Potassium
HERG
Cardiac delayed rectifier (IKr)
Dominant-negative loss of function with reduced repolarizing current
Episodic cardiac arrhythmias (LOT)
KVLQTI
? Cardiac potassium channel
? Reduced repolarizin9 current
Episodic cardiac arrhythmias (LOT)
KCNA 1
Neuronaldelayed rectifier
Dominant-negative, loss of function and reduced repolarizing current
Episodic ataxia
Chloride
CLC-I
Skeletal muscle chloride channel
Dominant-negative, loss of function and reduced repolartzing current
Episodic myotonia (myotoniacongenita)
Sodium
SCN4A
Skeletalmuscle sodium channel
Gain of function with increased depolarizing current
Episodic myotonia/periodic paralysis (hyperkalemic periodic paralysis)
SCN5A
Cardiac sodium channel
Gain of function with increased depolarizing current
Episodic cardiac arrythmia (LOT)
SCN8A
Neuronalsodium channel
Loss of channel expression leading to neuronal degradation
Paralysis, atrophy, Purkinje cell degeneration and death (motor endplate disease)
CACNLIA3
Skeletalmuscle dihydropydrine receptor
? Gain of function with increased depolarizing currents
Episodic myotonia/periodic paralysis (hypokalemia periodic paralysis)
13.ENaC
Amilodde-sensitive epithelial sodium channel
Increased sodium resorption
Hypertension (Liddle syndrome)
y-ENaC
Amiloride-sensltive epithelial sodium channel
Increased sodium resorption
Hypertension (Liddle syndrome)
MEC
? ligand-gated sodium c h a n n e l
Mechanosensory neurodegeneration
Touch-receptor neuronal degeneration in Caenorhabditis elegans
CFTR
Cystic fibrosis transmembrane conductance regulator
Loss of function with reduced sodium and chloride secretion
Cystic fibrosis
GLRA 1
Inhibitoryglycine receptor
?
Starlle disease
Water
AQP-2
Aquaporin-2water channel
Loss of function with reduced water resorption
Nephrogenic diabetes insipidus
Cation
ChRNA-4
Neuronalnicotinic acetylcholine receptor 0~4 receptor subunit
?
Epilepsy
deg-3
Nicotinic acetylcholine receptor
? Gain of function leading to channel hyperactivity
Neuronal degeneration in C. elegans
Girk2
Neuronalinward rectifier
Failed neuronal differentiation
Severe ataxia in mice
Calcium
Ligand-gated Sodium
Chloride
G protein coupled Potassium
Mechanism
Phenotype
CFTR, cystic fibrosis transmembrane conductance receptor; HERG, human ether-a-go-go; LOT, long CIT.
shown that mutations in the aquaporin-2 water channel gene, AQP-2, can cause an inherited form of nephrogenic diabetes insipidus [34"',35",36",37]. Expression of mutant channels in Xenopusoocytes indicates that these mutations result in a loss of function, leading to a reduction in the kidney's ability to concentrate urine. Protein trafficking studies suggest that these mutations retard processing of aquaporin-2 proteins to the plasma membrane [36"], leading to the quantitative reduction in functional water channels. The net result is a reduction in the kidney's ability to concentrate urine leading to polydipsia and polyuria in diabetes insipidus.
The cystic fibrosis transmembrane conductance regulator It has been known since 1988 that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are the cause of cystic fibrosis, but important work on the function of CFTR, and the functional consequences of CFTR-related mutations, are ongoing. CFTR is a chloride channel that requires ATP hydrolysis for gating. Electrophysiological studies have indicated that transition of CFTR between the closed and an open state depends on ATP binding, and that transition to a second open state requires ATP hydrolysis [38"].
330 Geneticsof disease
In addition to its function as a chloride channel, however, C F T R is known to regulate additional chloride and sodium transport pathways. Electrophysiological analyses of mutant and wild-type C F T R transfected into cultured airway epithelial cells indicate that C F T R regulates outwardly rectifying chloride channels. Extracellular ATP stimulates opening of these distinct, non C F T R , chloride channels [39°,40]. Mutant channels have lost both of these functional capabilities. Most mutations in C F T R that are associated with cystic fibrosis interfere with the biosynthesis of this channel, leading to rapid degradation before the molecules leave the endoplasmic reticulum. Two recent studies have focused on the mechanism through which C F T R s are targeted for degradation [41*,42"]. These studies have shown that several proteolytic systems, including the ubiquitin-proteasome pathway, contribute to C F T R degradation.
Disorders of ligand-gated channels Acetylcholine receptors T h e acetylcholine receptor (AChR) is a ligand-gated cation channel that is critical for synaptic transmission at the neuromuscular junction. It has been known for many years that dysfunction of AChRs is responsible for myasthenia gravis. In recent experiments, molecular genetic analyses of congenital myasthenic syndromes have indicated that missense mutations of the et subunits of the AChR can cause an inherited form of myasthenia [43",44,45]. Electrophysiological analyses of mutant AChR expressed in fibroblasts indicated a markedly decreased rate of acetylcholine disassociation, causing repeated channel openings. Recently, a missense mutation in the gene encoding the neuronal nicotinic AChR et4 subunit, CHRNA4, was linked to an inherited epileptic disorder known as autosomal dominant nocturnal frontal lobe epilepsy, indicating that these channels may also be involved in seizure disorders [46°]. T h e cellular mechanism of this disorder is not yet known.
Inhibitory glycine receptor T h e inhibitory glycine receptor is a hetero-oligomeric ligand-gated chloride channel that mediates inhibitory neurotransmission in the spinal cord and brain. T h e receptor is antagonized by the plant alkaloid strychnine. Reduced glycinergic inhibition caused by subconvulsive strychnine poisoning results in skeletal muscle hypertonia, increased reflex excitability, and exaggerated response to sensory stimuli. T h e adult isoform is composed of two integral membrane proteins, an ct and a [3 subunit. T h e ot I subunit is thought to contain the functional glycine receptor with four membrane-spanning segments each with extracellular amino and carboxyl termini. Recent linkage and mutational analyses have identified missense mutations in individuals with hereditary hyperekplexia
[47,48°]. This disorder, which is also known as familial Startle disease, is an autosomal dominant neurological disorder characterized by muscle rigidity of central nervous system origin and exaggerated startle response. The missense mutation identified in affected individuals is located near the putative ion channel region of the receptor and was predicted to affect chloride permeation. Physiological studies, however, have demonstrated that these mutations dramatically reduce receptor sensitivity to glycine [47,48"].
Neuronal differentiation and degeneration Inwardly rectifying potassium channel ot subunits are thought to form a tetrameric channel, the opening of which is coupled to activation of plasma membrane receptors via G proteins. These channels contain two membrane-spanning domains. T h e function of these channels is to maintain the resting membrane potential near the K+ equilibrium potential. Recent molecular genetic studies have shown that mutations in a neuronal inward rectifying potassium channel gene, Girk2, cause the re,eaver phenotype in mice [49"]. Mice which are homozygous for the re,eaver mutation suffer from severe ataxia that is apparent by the second post-natal week. T h e precise mechanism for the aseaver phenotype is not clear, but is thought to involve failed differentiation ofcerebellar neurons, specifically granule cells. These data suggest that the hyperpolarization mediated by Girk2 channels is essential for cerebellar neuronal differentiation. Recent experiments have also led to the implication of ion channel function in neurodegeneration. Genetic studies in the nematode Caenorhabditis elegans have defined a sct of genes known as degenenns that arc required for the function of touch-receptor neurons [50"°]. These genes share a considerable DNA sequence similarity to the amiloride-sensitive sodium channel. These data suggest, therefore, that dysfunction of neuronal ion channels in C. elegans can cause neurodegcneration. Another C. elegans mutant has also implicated ion channel mutation in neurodegeneration. The deg-3 gene encodes a nicotinic AChR ct subunit. A mutation in this gene causes degeneration of a small set of neurons in (7. elegans [51"]. Although the mechanism has not been tested specificall% a similar mutation in the chick protein produces channels that desensitize slowly. Additional evidence that channel hyperactivity is the mechanism for this phenomenon is indicated by the fact that a nicotinic receptor antagonist ameliorated the phenotype. Molecular genetic studies in mice have also implicated voltage-gated sodium channels in neurodegeneration. A mouse mutant known as 'motor endplate disease' [52"] is characterized by progressive paralysis, severe muscle atrophy, degeneration of Purkinje cells and death. A mutation in the voltage-gated sodium channel gene, SCNSA, has been implicated in this disorder [52°]; the
Pethophysiology of ion channel mutations Keating and Sanguinetti
331
mutation leads to a loss of SCN8A expression. As this is a recessive diso/der, these data indicate that a loss of SCN8A function contributes to neuronal degeneration.
delayed rectifier potassium current (IKr) in cardiac myocytes. This established HERG as the gene encoding cc subunits that form the IKr potassium channel. Because block of IKr is a known mechanism for drug-induced cardiac arrhythmias, the finding that HERG encodes IKr channels provided a mechanistic link between inherited and acquired cardiac arrhythmias.
Conclusions
5.
Trudeau M, Warmke J, Ganetzky B, Robertson G: HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 1995, 269:92-95.
6.
Spector P, Curran M, Keating M, Banguinetti M: Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Circ Res 1996, 78:499-603.
Mutations in voltage-gated potassium and chloride channels have been implicated in several muscle and neural disorders. These mutations are thought to cause abnormal cellular repolarization and increase cellular excitability. Depending on the organ involved, these inherited disorders can lead to cardiac arrhythmias, myotonia, or ataxia. In addition, it is now clear that dysfunction of ion channels can contribute to abnormal neural differentiation and neural degeneration. It seems likely that inherited and acquired dysfunction of ion channels will eventually prove important in a large number of disorders involving excitable and nonexcitablc cells.
Note added in proof Mutations in 6"LCNS, the gene encoding a chloride channel expressed in the kidney, cause three types of inherited hypercalciuric nephrolithiasis [53]. These mutations lead to the loss, or greatly diminish the function of, channels expressed in Xenopusoocytes. It is not known where in the kidney this channel is expressed or how mutations in the protein alter resorption of calcium, leading to the formation of kidney stones.
7 •
Sanguinetti MC, Curran ME, Spector P and Keating MT: Spectrum of HERG K+ channel dysfunction in an inherited cardiac arrhythmia. Proc Nat/Acad Sci USA 1996, 93:2208-2212. Here, we demonstrated that LQT-associated mutations in HERG cause loss, alteration and dominant-negative suppression of HERG function. Reduced repolarizing HERG potassium current presumably leads to increased myocellular excitability and arrhythmia susceptibility. 8. •.
Browne D, Gancher S, Nutt J, Brunt E, Smith E, Kramer P, Litt M: Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNAI. Nat Genet 1994, 8:136-140. This manuscript was the first to demonstrate that mutations in the human brain potassium channel gene, KCNA 1, cause episodic ataxia and myokymia (muscle rippling). 9.
10. •
Adelman J, Bond C, Pessia M, Maylie J: Episodic ataxia results from voltage-dependent potassium channels with altered functions. Neuron 1995, 15:1449-1454. This manuscript showed that mutations in the voltage-dependent delayed rectifier potassium channel gene, KCNA1, associated with episodic ataxia cause loss, alteration and dominant-negative suppression of channel funclion. 11.
George A, Crackower M, Abdalla J, Hudson A, Ebers G: Molecular basis of Thomsen's disease (autosornal dominant myotonia congenita). Nat Genet 1993, 3:305-310.
1 2.
Steinmeyer K, Lorenz C, Pusch M, Koch M, Jentsch T: Multimeric structure of CLC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen). EMBO J 1994, 13:737-743.
13.
Gronemeier M, Condie A, Prosser J, Steinmeyer K, Jentsch T, Jockusch H: Nonsense and missense mutations in the muscular chloride channel gene CLC-1 of myotonic mice. J Big~ Chem 1994, 269:5963-5967.
14.
Heine R, George A, Pika U, Deymeer F, Rudel R, Lehmann-Horn F: Proof of a non-functional muscle chloride channel in recessive myotonia congenita (Becker) by detection of a 4 base pair deletion. Hum Mo/Genet 1994, 3:1123-1128.
16.
Lorenz C, Meyer-Kleine C, Steinmeyer K, Koch M, Jentsch T: Genomic organization of the human muscle chloride channel CIC-1 and analysis of novel mutations leading to Becker-type myotonia. Hum Mol Genet 1994, 3:941-946.
16.
George A, Sloan-Brown K, Fenichel G, Mitchell G, Spiegel R, Pascuzzi R: Nonsense and missense mutations of the muscle chloride channel gene in patients with myotonia congenita. Hum Mo/ Genet 1994, 3:2071-2072.
Acknowledgements T h e authors thank H Katchuk,
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.
of special interest of outstanding interest Jiang C, Atkinson D, Towbin JA, Lehmann M, Splawski I, Li H, Taggart RT, Timothy K, Schwartz PJ, Vincent GM, Moss AJ, Keating MT: Two long QT syndrome loci map to chromosomes 3 and 7 with evidence for further heterogeneity. Nat Genet 1994, 8:141-147.
2. ••
Curran ME, Splawski I, Timothy K, Vincent GM, Green E, Keating MT: A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Ceil 1995, 80:795-803. This manuscript demonstrated that mutations in HERG, a putative potassium channel gene, cause an inherited cardiac arrhythmia, LQT. The discovery was the first to demonstrate that mutations in cardiac ion channel genes cause susceptibility to life-threatening arrhythmias. 3. ••
Wang Q, Curran M, Splawski I, Burn T, Millholland J, VanRaay T, Shen J, Timothy K, Vincent GM, De Jager T et al.: Positional cloning of a novel cardiac channel gene: KVLQT1 mutations cause cardiac arrhythmias, Nat Genet 1996, 12:1-23. In this manuscript, positional cloning techniques were used to identify a novel cardiac potassium channel gene, KVLQT1, and demonstrate that mutations in this gene cause the most common form of inherited LOT syndrome. KVLQT1 is predicted to be a voltage-gated potassium channel important for myocellular repolarization. LQT-associated mutations were predicted to have a dominant-negative effect on KVLGT1 function. 4. =•
Sanguinetti MC, Jiang C, Curran ME, Keating MT: A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Ceil 1995, 81:299-307 In this manuscript, we demonstrated that the biophysical properties of HERG expressed in Xenopus oocytes were nearly identical to the rapidly activating
Browne D, Brunt E, Griggs R, Nutt J, Gancher S, Smith S, Litt M: Identification of two new KCNA1 mutations in episodic ataxia/myokymia families. Hum Mol Genet 1995, 4:1671-16?2.
17 •
Pusch M, Steinmeyer K, Koch M, Jentsch T: Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CLC-1 chloride channel. Neuron 1995, 15:1455-1463, This manuscript provides the first demonstration that mutations in the skeletal muscle chloride channel, CLC-1, cause a dramatic shift in the voltage sensitivity of expressed channels, leading to a loss of function. These mutant channels cannot contribute to repolarization of skeletal muscle action potential, leading to myotonia. 18. •.
Wang Q, Shen J, Splawski I, Atkinson DL, Li Z, Robinson J, Moss A, Towbin J, Keating MT: SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome, Ceil 1995, 80:805-811. This work was the first to demonstrate that mutations in the cardiac sodium channel gene SCN5A cause LQT syndrome. These findings suggested that the cellular mechanism of the disease results from altered channel inactivation properties and continued depolarizing sodium current late in the plateau phase of the cardiac action potential.
332
Genetics of disease
19.
Wang Q, Shen J, Li Z, Timothy K, Vincent GM, Priori S, Schwartz P, Keating MT: Cardiac sodium channel mutations in patients with long QT syndrome, an inherited cardiac arrhythmia. Hum Mol Genet 1995, 4:1603-1607.
20. Bennett P, Yazawa K, Makita N, George A: Molecular mechanism • for an inherited cardiac srrhythmia. Nature 1995, 376:683-685. This manuscript was the first to demonstrate that LQT-associated mutations in the cardiac sodium channel gene cause destabilization of the channel's inactivation gate and late channel reopenings. 21.
Hoffman E, Lehmann-Horn F, Rudel R: Overexcited or inactive: Ion channels in muscle disease. Cell 1995, 80:681-686.
22.
Yang N, Zhou M, Ptacak L, Barchi R, Horn R, George F: Sodium channel mutations in paramyotonia congenita exhibit similar biophysical phenotypes in vitro. Proc Nat/Acad Sci USA 1994, 91:12785-12789.
23.
Zhou .I, Spier S, Beech J, Hoffman E: Pathophysiology of sodium channelopathles: con'elatlon of normal/mutant mRNA ratios with clinical phenotype in dominantly inherited periodic paralysis. Hum Mol Genet 1994, 3:1599-1603.
24. •-
Ptacek L, Tawil R, Griggs R, Engel A, Layzer R, Kwiecinski H, McManis P, Santiago L, Moore M, Fouad G e t aL: Dlhydropydrine receptor mutations cause hypokalemic periodic paralysis. Cell 1994, 77:863-868. This is the first demonstration that mutations in the skeletal muscle voltage gated calcium channel gene (also known as the dihydropydrine receptor gene) cause episodic weakness associated with low serum potassium levels- hypokalemic periodic paralysis. 25. ••
Jurkat-Rott K, Lehmann-Horn F, Elbaz A, Heine R, Gregg R, Hogan K, Powers P, Lapie P, Vale-Santos J, Weissenbach J, Fontaine B: A calcium channel mutation causing hypokalemlc periodic paralysis. Hum Mol Genet 1994, 3:141 5-141 g. This manuscript established that mutations in the dihydropydrine receptor gene cause hypokalemic periodic paralysis. 26.
Sipos I, Jurkatt-Rott K, Harasztosi Cs, Fontaine B, Kovacs L, Melzer W, Lehmann-Horn F: Skeletal muscle DHP receptor mutations alter calcium currents in human hypokalaemic periodic paralysis myotubes. J Physio11995, 483:299-306.
27. ••
Canessa C, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J-D, Rossier B: Amlloride-sensitlve epithelial Na+ channel is made of three homologous subunits. Nature 1994, 367:483-46?. This study defined the primary structure of the genes that encode the epithelial sodium channel, demonstrating that it comprises three homologous subunits that interact to form functional channels. 28.
Snyder P, McDonald F, Stokes J, Welsh M: Membrane topology of the smiloride-sensitive epithelial sodium channel. J Bio/ Chem 1994, 269:24379-24383.
29. • ~,
Shimkets R, Warnock D, Bositis C, Nelson-Williams C, Hansson J, Schambelan M, Gill J, Ulick S, Milora R, Finding Jet al.: Liddle's syndrome: heritable human hypertension caused by mutations in the I~ subunit of the epithelial sodium channel. Cell 1994, 79:407-414. Here, the authors demonstrate that mutations in the ~ subunit of the epithelial sodium channel cause Liddle syndrome (pseudo aldosteronism), an autosoreal dominant form of human hypertension. The data presented suggest that the mechanism of Liddle syndrome is constitutive activation of an amiloridesensitive distal nephronal epithelial sodium channel. These mutations lead to increased sodium resorption by the kidney and susceptibility to hypertension. 30. •
Hansson J, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton R: Hypertension caused by a truncated epithelial sodium channel 7 subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 1995, 11:76-82. This manuscript showed that mutations in the Y subunit of the epithelial sodium channel cause susceptibility to hypertension in Liddle syndrome. 31. •
Schild L, Caness C, Shimkets R, Gautschi I, Lifion R, Rossier B: A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Nat/Acad Sci USA 1995, 92:569g-5703. In this manuscript, the authors expressed Liddle syndrome associated mutant 13subunits of the epithelial sodium channel with wild-type ~ and y subunits in Xenopus oocyles, demonstrating an increase in the macroscopic amiloride-sensitive sodium current. 32. •
Snyder P, Price M, McDonald F, Adams C, Volk K, Zeiher B, Stokes J, Welsh M: Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel. Cell 1995, 83:969-978.
This is the first demonstration that Liddle syndrome associated mutations in the epithelial sodium channel genes cause an increase in the number of sodium channels in the apical membrane and do not alter single-channel conductance or open-state probability. 33.
Jung J, Preston G, Smith B, Guggino W, Agre P: Molecular structure of the water channel through aquaporin CHIP. J Biol Chem 1994, 269:14648-14654.
34. •-
Deen P, Verdijk M, Knoers N, Wieringa B, Monnens L, Van Os C, Van Oost B: Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science 1994, 264:92-95. This was the first demonstration that the aquaporin-2 water channel was required for concentration of urine by the kidney. These findings suggested that mutations in the aquaporin-2 water channet gene might cause diabetes insipidus. Support for this hypothesis was demonstrated in one patient. 35. •
Van Lieburg A, Verdijk M, Knoers V, Van Essen A, Poresmans W, Mallmann R, Monnens L, Van Oost B, Van Os C, Deen P: Patients with autosomal nephrogenic diabetes insipidus homozygous for mutations in the aquaporin 2 water-channel gene. Am J Hum Genet 1994, 55:648-652. This manuscript identified missense mutations and a single nucleotide deletion in the aquaporin 2 gene in three individuals with nephrogenic diabetes insipidus. Expression of the mutants in Xenopus oocytes showed that the resultant channels were nonfunctional. These data indicate that mutations in the aquaporin-2 water channel gene can cause autosomal recessive nephrogenic diabetes insipidus. 36. •
Deen P, Croes H, Van Aubel R, Ginsel L, Van Os C: Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes instpidus are impaired in their cellular routing. J Ch'n Invest 1995, 95:2291-2296. This study presented immunolocalization data suggesting that diseaseassociated mutant forms of the aquaporin-2 water channel proteins fail to reach the plasma membrane, leading to nonfunctional water channels. 37.
Preston G, Smith B, Zeidel M, Moulds J, Agre P: Mutations in aquaporin-1 in phenofypically normal humans without functional CHIP water channels. Science 1994, 265:1585-1587.
38. •
Gunderson K, Kopito R: Conformational states of CFTR associated with channel gating: the role of ATP binding and hydrolysis. Cell 1995, 82:231-239. In this manuscript, electrophysiologic analyses of wild-type and mutant CFTR channels indicated that binding of ATP causes a reversible transition from the closed to an open state. Transition from this open state requires ATP hydrolysis. 39. •
Schwiebert E, Egan M, Hwang T, Fulmer S, Allen S, Cutting G, Guggino W: CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATR Ceil 1995, 81:1063-1073. Physiological studies have suggested that CFTR can interact to modulate outwardly rectifying chloride channels in addition to being a chloride channel itself. Data provided in this manuscript indicated that CFTR regulates outwardly rectifying chloride channels by triggering the transport and release of ATP. ATP, in turn, activates non-CFTR chloride channels. 40.
Reisin I, Prat A, Abraham E, Amara J, Gregory R, Ausiello D, Cantiello H: The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J Biol Chem 1994, 269:20584-20591.
41. •
Jensen 1", Loo M, Pind S, Williams D, Goldberg A, Riordan J: Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 1995, 83:129-135. CFTR levels are tightly regulated, in part by protein degradation. Proper trafficking in early degradation of mutant CFTR proteins is thought to be mechanistically important in cystic fibrosis. This manuscript demonstrated that the molecular mechanisms of CFTR processing include degradation in the proteasome. 42. Ward C, Omura S, Kopito R: Degradation of CFTR by the • ubiquitin-proteasome pathway. Cell 1995, 83:121-127. This manuscript demonstrated that the ubiquitin-proteasome pathway is important for CFTR degradation. 43. •
Sine S, Ohno K, Bouzat C, Auerbach A, Milone M, Pruitt J, Engel A: Mutation of the acetylcholine receptor ~ subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity. Neuron 1995, 15:229-239. This study demonstrated that mutations in the AChR ct subunit cause slowchannel myasthenic syndrome in several individuals. Electrophysiological analysis revealed a decreased rate of acetylcholine disassociation, causing the mutant receptors to open repeatedly during ligand occupancy, thereby altering the synaptic response. 44.
Ohno K, Hutchinson D, Milone M, Brengman J, Bouzat C, Sine S, Engel A: Congenital myasthenic syndrome caused by
P a t h o p h y s i o l o g y o f ion channel mutations Keating and Sanguinetti
prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the E subuniL Proc Nat/Acad Sci USA 1995, 92:758-762. 45.
Gomez C, Gammack J: A leucine-to-phenylalanine substitution in the acetylcholine receptor ion channel in a family with the slow-channel syndrome. Neuro/ogy 1995, 45:982-985.
46. •
Steinlein O, Mulley J, Propping P, Wallace R, Phillips H, Sutherland G, Scheffer I, Berkovic S: A missense mutation in the neuronal nicotinic acetylcholine receptor c¢4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 1995, 11:201-203. This study presented data suggesting that a missense mutation in the neuronal nicotinic AChR may be the cause of autosomal dominant nocturnal frontal lobe epilepsy in humans. The functional consequences of this mutant are not yet known. 47.
Shiang R, Ryan S, Zhu Y-Z, Hahn A, O'Connell P, Wasmuth J: Mutations in the c~1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nat Genet 1993, 5:351-358.
48. •
Rajendra S, Lynch J, Pierce K, French C, Barry P, Schofield P: Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor. J Bio/Chem 1994, 269:18739-18742. This manuscript demonstrated that mutations in the glycine receptor, a ligand-gated chloride channel associated with Startle disease, disrupt receptor function by causing a dramatic decrease in sensitivity to glycine. Startle disease mutations in the glycine receptor reduce the efficiency of glycinergic inhibitory neurotransmission. 49. •.
Patil N, Cox D, Bhat E), Faham M, Myers R, Peterson A: A potassium channel mutation in weaver mice implicates
333
membrane excitability in granule cell differentiation. Nat Genet 1995, 11:126-129. This manuscript demonstrated that a missense mutation in a G-protein-coupled inward rectifier potassium channel gene cause the weaver phenotype in mice. Homozygous animals suffer from severe ataxia that is thought to result from a block in neuronal differentiation in the cerebellum. 50. •o
Huang M, Chalfie M: Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans. Nature 1994, 367:467-470. This study characterizes two genes in C. elegans that are critical for mechanosensory transduction, and, from DNA sequence homology, are similar to the amiloride-sensitive sodium channel c~, 13and ~, subunits. 51. •
Treinin M, Chalfie M: A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans. Neuron 1995, 14:871-877. This manuscript demonstrated that a gain-of-function mutation in an AChR subunit leads to degeneration of a set of neurons in C. elegans. Because antagonists of nicotinic AChRs suppress the mutant phenotype, these data suggest that channel hyperactivity may underlie neurodegeneration in this model. 52. •
Burgess D, Kohrman D, Gait J, Plummer N, Jones J, Spear B, Meisler M: Mutation of a new sodium channel gene, ScnSe, in the mouse mutant 'motor endplate disease'. Nat Genet 1995, 10:461-465. This manuscript demonstrated that mutation of a brain sodium channel gene is responsible for neurodegeneration in the mouse mutant known as 'motor endplate disease'. 53.
Lloyd SE, Pearce SHS, Fisher SE, Steinmeyer K, Schwappach B, Scheinman S.I, Harding B, Brolino A, Devoto M, Goodyer Pet al: A common molecular basis for three inherited kidney stone diseases. Nature 1996, 379:445-449.
Pathophysiology of ion channel mutations Mark T Keating* and Michael C Sanguinettit The past year has seen significant advances in our understanding of ion channel disorders. The highlights of these advances include a detailed delineation of the molecular mechanisms underlying inherited cardiac arrhythmias and the discovery that ion channel mutations can contribute to neural development and neurodegeneration.
Address *Howard Hughes Medical Institute, University of Utah, Building 533, Room 6110B, Salt Lake City, Utah 84112, USA
tEccles Program in Human Molecular Biology and Genetics, University of Utah, Building 533, Room 4220, Salt Lake City, Utah 84112, USA
Current Opinion in Genetics & Development 1996, 6:326-333 © Current Biology Ltd ISSN 0959-437X
Abbreviations HERG
LQT CFTR
AChR
humanether-a-go-go related gene long QT cystic fibrosis transmembrane conductance regulator acetylcholine receptor
Introduction Ion channels regulate a wide variety of cellular functiolas. Not surprisingly, it has become clear that mutations in genes encoding ion channels contribute to a wide variety of pathological disorders in both excitable and non-excitable cells. These conditions include disorders of cellular excitability in the heart (long Q T [LQT] syndrome, an inherited cardiac arrhythmia), skeletal muscle (myotonia and periodic paralysis), and the central nervous system (episodic ataxia and seizures). Mutations and genes encoding ion channels also contribute to disorders of fluid and electrolyte metabolism, including hypertension and nephrogenic diabetes insipidus. Ion channel dysfunction can also contribute to neural development and degeneration. T h e two major discoveries in the past year were the discovery of the molecular mechanisms underlying life-threatening cardiac arrhythmias and the role of ion channels in neural development and degeneration. Voltage-gated ion channel mutations and cellular excitability Loss, alteration, and dominant-negative suppression of potassium and chloride channel function can lead to delayed cellular repolarization and increased excitability Voltage-gated potassium channels comprise a large family of proteins with varying functions and distinct structural motifs. A common class of potassium channel c~ subunits are thought to contain six membrane-spanning segments, SI-S 6, and a pore-forming domain located between S 5 and $6 (Fig. 1). T h e fourth membrane-spanning segment, $4, contains a positively charged amino acid residue at every
third position of the putative cc helix and is thought to be critical for voltage sensitivity. As functional potassium channels are formed from the co-assembly of four o~ subunits, either as homotetramers or heterotetramers, some mutations may have a dominant-negative effect. L Q T syndrome is an inherited disorder that causes syncope and sudden death in young, otherwise healthy, individuals. T h e symptoms result from episodic cardiac arrhythmias, specifically torsade de pointes and vcntricular fibrillation. T h e only other phenotypic abnormality in affected individuals is subtle and variable prolongation of the Q T interval on surface electrocardiograms, an indication of abnormal cardiac repolarization. Molecular genetic studies of familial and sporadic cases have demonstrated that missense mutations and small deletions in cardiac voltage-gated potassium channel genes can lead to this disorder. A positional/candidate gene approach was used to demonstrate that mutations in the human ether-a-go-go related gene (HERG; Table 1) cause chromosome 7 linked L Q T [1,2*'], whereas a pure positional cloning approach was used to identify a novel potassium channel gene, KVLQT1, and demonstrate that mutations in this gene cause the chromosome 11 linked form of this disorder [3°°]. Heterologous expression of HERG in Xenopus oocytes led to the discovery that this gene encodes subunits which form a cardiac delayed rectifier potassium channel, IKr [4°°]. T h e biophysical and pharmacological properties of HERG homotetramers expressed in oocytes appear nearly identical to those of IKr in cardiac myocytes [5,6]. Previous studies have indicated that a pharmacological block of IKr is a common cause of drug-induced arrhythmias, providing a mechanistic link between inherited and acquired cardiac arrhythmias. Co-expression of mutant and wild-type H E R G channels has led to the discovery that most LQT-associated mutations cause dominant-negative loss of H E R G function [7°]. T h e potency of the dominant-negative effect is variable; some mutants have relatively little or no effect on normal H E R G subunits, whereas others lead to a near total disruption of t I E R G function. Thus, these mutations lead to a spectrum of H E R G sodium channel dysfunction in LQT. The function of the protein encoded by KVLQT1 is not yet known, but it is likely to be a voltage-gated potassium channel which is important for myocellular repolarization. These data are consistent with the hypothesis that I , Q T results from delayed myocellular repolarization, secondary depolarizations (afterdepolarizations), and increased risk of cardiac arrhythmia. A similar mechanism has been implicated in a disorder of the central nervous system--episodic ataxia/myokymia
Pathophysiology of ion channel mutations Keating and Sanguinetti 32?
Figure 1
(a)
Potassium $1
S2
S3
S4
S6 Extracellular
rCOOH_ (b) Chloride
(c)
4
COOH-
Sodium/Calcium DI
DII
Dill
DIV Extracellular
l NH3+
Intracellular
~ C O O H ,~"1996CurrentOpinioninGenetics&Development
Predicted topology for (z subunits of voltage-gated ion channels: (a) potassium, (b) chloride and (c) sodium/calcium channels. Most functional potassium channels are formed by the coassembly of four identical subunits, analagous to a single sodium or calcium c~subunit. The 'barrels' (e.g. S1-S 6 in the potassium channel) represent (z helices. The pores of these channels in (a) and (b) are formed by the loops between segments $5 and $6; the $4 transmembrane region is thought to be the voltage sensor. The topology of the chloride channel subunits is uncertain, but functional chloride channels are probably formed by the coassembly of 4 subunits.
328 Genetics of disease
syndrome. In this disorder, individuals suffer from episodic bouts of severe ataxia and mild myokymia (muscle rippling). Molecular genetic studies have implicated missense mutations in KCNA1, a voltage-gated potassium channel gene expressed in cerebellar neurons [8"*,9]. Physiological studies indicate that disease-associated mutations in KCNA1 lead to altered channel function (e.g. faster inactivation or altered voltage dependence of activation) and dominant-negative suppression of repolarizing K + current [10"]. Potassium channel mutations have not yet been implicated in a disorder of skeletal muscle, but a similar mechanism is thought to be involved in the pathogenesis of myotonia congenita, a disorder that causes episodic muscle spasms. Molecular genetic studies have shown that this disorder results from mutations in a skeletal muscle chloride channel gene, CLC1 [11-16]. Chloride channels are also thought to form oligomers and physiological studies indicate that these mutations cause dominant-negative suppression of chloride current [17"]. One of the more exciting aspects of these studies has been their therapeutic implications. It is likely, for example, that the risk of arrhythmia in an individual with mutations of HERG or KVLQTI can be reduced with potassium channel opening agents or even supplementation with potassium itself. H E R G is paradoxically sensitive to extracellular potassium concentration; increasing extracellular potassium in a physiological range increases outward potassium current through H E R G channels [4"*]. The precise mechanism of this phenomenon is not known, but probably involves allosteric interactions between potassium ions and the extracellular domain of the channel, resulting in an increase in open probability. Preliminary data suggest that modest increases in serum potassium concentration correct cardiac repolarization abnormalities in patients with chromosome 7 linked LQT. This therapeutic approach should also work for patients with drug-induced L Q T and may be effective for L Q T resulting from mutations in other genes (KVLQT1, SCNSA, etc.). Gain of function mutations in sodium channel genes can cause abnormal cellular repolarization and increased excitability Activation of voltage-gated sodium channels initiates the action potential in muscle and neurons. T h e ct subunit of these channels is thought to contain four major domains (DI-DIV), each of which is thought to contain six membrane-spanning segments. A single sodium channel ct subunit is sufficient for channel function, but some functional characteristics arc modulated by other subunits.
Molecular genetic studies have shown that missense mutations and small deletions of the cardiac sodium channel gene SCN5A are a rare cause of inherited L Q T [18*',19]. T h e mutations that have been identified to date have been in the interdomain segment between D i l l and DI'v:
This region is important for sodium channel inactivation. Expression of mutant sodium channels in Xenopus oocytes, however, did not show significant changes in the kinetics of channel inactivation. Instead, a subtle maintained sodium current was detected. Single channel recordings indicated infrequent late reopenings, leading to depolarizing inward current late in the action potential [20*]. Thus, LQT-associated mutations in SCN5A destabilize the channel's inactivation gate. Continued depolarizing sodium current late in the plateau phase of the myocellular action potential presumably prolongs action potential duration, increasing the risk of afterdepolarizations and arrhythmia. A similar mechanism was previously demonstrated for hyperkalemic periodic paralysis, a disorder of skeletal muscle. This disorder results from gain of function mutations in a skeletal muscle sodium channel gene leading to episodic skeletal muscle excitation (myotonia) and periodic paralysis [21-23]. Voltage-gated calcium channel ct subunits have a structure that is similar to sodium channel et subunits. Molecular genetic studies have implicated missense mutations in voltage-gated L-type calcium channel ct subunits in the skeletal muscle disorder hypokalemic periodic paralysis [24*',25"',26]. In this disorder, individuals suffer from episodic myotonia followed by paralysis in the setting of low serum potassium concentrations. Although the precise functional consequences of these mutations are not clear, it is likely that these mutations increase inward calcium current and prolong cellular repolarization. Ion c h a n n e l m u t a t i o n s a f f e c t i n g w a t e r a n d electrolyte homeostasis Epithelial sodium channels and hypertension T h e epithelial sodium channel is expressed in the kidney and colon and is critical for sodium resorption. T h e channel comprises ~, 13 and T subunits and is activated by angiotensin [27"*,28]. Molecular genetic studies have shown that mutations in epithelial sodium channel genes cause Liddle syndrome, a rare inherited disorder that causes salt-sensitive hypertension [29**,30*]. Physiological studies of mutant channel subunits indicate an increase in sodium absorption [31",32"]. Surprisingly, the mutant mechanism does not appear to involve altered biophysical properties; instead, the molecular mechanism appears to be increased numbers of epithelial sodium channel subunits, probably caused by altered protein degradation. The aquaporin-2 water channel and diabetes insipidus T h e aquaporin-2 water channel has a topology similar to potassium channels; the predicted structure suggests that the amino and carboxyl termini are located in the intracellular compartment and the protein has six putative membrane-spanning domains [33]. These channels are known to be important for vasopressin-induced resorption of water by the kidney. Molecular genetic studies have
Pathophysiology of ion channel mutations Keating and Sanguinetti 329
Table 1 Pathophysiology of ion channel mutations. Channels
Gene
Protein
Voltage-gated Potassium
HERG
Cardiac delayed rectifier (IKr)
Dominant-negative loss of function with reduced repolarizing current
Episodic cardiac arrhythmias (LOT)
KVLQTI
? Cardiac potassium channel
? Reduced repolarizin9 current
Episodic cardiac arrhythmias (LOT)
KCNA 1
Neuronaldelayed rectifier
Dominant-negative, loss of function and reduced repolarizing current
Episodic ataxia
Chloride
CLC-I
Skeletal muscle chloride channel
Dominant-negative, loss of function and reduced repolartzing current
Episodic myotonia (myotoniacongenita)
Sodium
SCN4A
Skeletalmuscle sodium channel
Gain of function with increased depolarizing current
Episodic myotonia/periodic paralysis (hyperkalemic periodic paralysis)
SCN5A
Cardiac sodium channel
Gain of function with increased depolarizing current
Episodic cardiac arrythmia (LOT)
SCN8A
Neuronalsodium channel
Loss of channel expression leading to neuronal degradation
Paralysis, atrophy, Purkinje cell degeneration and death (motor endplate disease)
CACNLIA3
Skeletalmuscle dihydropydrine receptor
? Gain of function with increased depolarizing currents
Episodic myotonia/periodic paralysis (hypokalemia periodic paralysis)
13.ENaC
Amilodde-sensitive epithelial sodium channel
Increased sodium resorption
Hypertension (Liddle syndrome)
y-ENaC
Amiloride-sensltive epithelial sodium channel
Increased sodium resorption
Hypertension (Liddle syndrome)
MEC
? ligand-gated sodium c h a n n e l
Mechanosensory neurodegeneration
Touch-receptor neuronal degeneration in Caenorhabditis elegans
CFTR
Cystic fibrosis transmembrane conductance regulator
Loss of function with reduced sodium and chloride secretion
Cystic fibrosis
GLRA 1
Inhibitoryglycine receptor
?
Starlle disease
Water
AQP-2
Aquaporin-2water channel
Loss of function with reduced water resorption
Nephrogenic diabetes insipidus
Cation
ChRNA-4
Neuronalnicotinic acetylcholine receptor 0~4 receptor subunit
?
Epilepsy
deg-3
Nicotinic acetylcholine receptor
? Gain of function leading to channel hyperactivity
Neuronal degeneration in C. elegans
Girk2
Neuronalinward rectifier
Failed neuronal differentiation
Severe ataxia in mice
Calcium
Ligand-gated Sodium
Chloride
G protein coupled Potassium
Mechanism
Phenotype
CFTR, cystic fibrosis transmembrane conductance receptor; HERG, human ether-a-go-go; LOT, long CIT.
shown that mutations in the aquaporin-2 water channel gene, AQP-2, can cause an inherited form of nephrogenic diabetes insipidus [34"',35",36",37]. Expression of mutant channels in Xenopusoocytes indicates that these mutations result in a loss of function, leading to a reduction in the kidney's ability to concentrate urine. Protein trafficking studies suggest that these mutations retard processing of aquaporin-2 proteins to the plasma membrane [36"], leading to the quantitative reduction in functional water channels. The net result is a reduction in the kidney's ability to concentrate urine leading to polydipsia and polyuria in diabetes insipidus.
The cystic fibrosis transmembrane conductance regulator It has been known since 1988 that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are the cause of cystic fibrosis, but important work on the function of CFTR, and the functional consequences of CFTR-related mutations, are ongoing. CFTR is a chloride channel that requires ATP hydrolysis for gating. Electrophysiological studies have indicated that transition of CFTR between the closed and an open state depends on ATP binding, and that transition to a second open state requires ATP hydrolysis [38"].
330 Geneticsof disease
In addition to its function as a chloride channel, however, C F T R is known to regulate additional chloride and sodium transport pathways. Electrophysiological analyses of mutant and wild-type C F T R transfected into cultured airway epithelial cells indicate that C F T R regulates outwardly rectifying chloride channels. Extracellular ATP stimulates opening of these distinct, non C F T R , chloride channels [39°,40]. Mutant channels have lost both of these functional capabilities. Most mutations in C F T R that are associated with cystic fibrosis interfere with the biosynthesis of this channel, leading to rapid degradation before the molecules leave the endoplasmic reticulum. Two recent studies have focused on the mechanism through which C F T R s are targeted for degradation [41*,42"]. These studies have shown that several proteolytic systems, including the ubiquitin-proteasome pathway, contribute to C F T R degradation.
Disorders of ligand-gated channels Acetylcholine receptors T h e acetylcholine receptor (AChR) is a ligand-gated cation channel that is critical for synaptic transmission at the neuromuscular junction. It has been known for many years that dysfunction of AChRs is responsible for myasthenia gravis. In recent experiments, molecular genetic analyses of congenital myasthenic syndromes have indicated that missense mutations of the et subunits of the AChR can cause an inherited form of myasthenia [43",44,45]. Electrophysiological analyses of mutant AChR expressed in fibroblasts indicated a markedly decreased rate of acetylcholine disassociation, causing repeated channel openings. Recently, a missense mutation in the gene encoding the neuronal nicotinic AChR et4 subunit, CHRNA4, was linked to an inherited epileptic disorder known as autosomal dominant nocturnal frontal lobe epilepsy, indicating that these channels may also be involved in seizure disorders [46°]. T h e cellular mechanism of this disorder is not yet known.
Inhibitory glycine receptor T h e inhibitory glycine receptor is a hetero-oligomeric ligand-gated chloride channel that mediates inhibitory neurotransmission in the spinal cord and brain. T h e receptor is antagonized by the plant alkaloid strychnine. Reduced glycinergic inhibition caused by subconvulsive strychnine poisoning results in skeletal muscle hypertonia, increased reflex excitability, and exaggerated response to sensory stimuli. T h e adult isoform is composed of two integral membrane proteins, an ct and a [3 subunit. T h e ot I subunit is thought to contain the functional glycine receptor with four membrane-spanning segments each with extracellular amino and carboxyl termini. Recent linkage and mutational analyses have identified missense mutations in individuals with hereditary hyperekplexia
[47,48°]. This disorder, which is also known as familial Startle disease, is an autosomal dominant neurological disorder characterized by muscle rigidity of central nervous system origin and exaggerated startle response. The missense mutation identified in affected individuals is located near the putative ion channel region of the receptor and was predicted to affect chloride permeation. Physiological studies, however, have demonstrated that these mutations dramatically reduce receptor sensitivity to glycine [47,48"].
Neuronal differentiation and degeneration Inwardly rectifying potassium channel ot subunits are thought to form a tetrameric channel, the opening of which is coupled to activation of plasma membrane receptors via G proteins. These channels contain two membrane-spanning domains. T h e function of these channels is to maintain the resting membrane potential near the K+ equilibrium potential. Recent molecular genetic studies have shown that mutations in a neuronal inward rectifying potassium channel gene, Girk2, cause the re,eaver phenotype in mice [49"]. Mice which are homozygous for the re,eaver mutation suffer from severe ataxia that is apparent by the second post-natal week. T h e precise mechanism for the aseaver phenotype is not clear, but is thought to involve failed differentiation ofcerebellar neurons, specifically granule cells. These data suggest that the hyperpolarization mediated by Girk2 channels is essential for cerebellar neuronal differentiation. Recent experiments have also led to the implication of ion channel function in neurodegeneration. Genetic studies in the nematode Caenorhabditis elegans have defined a sct of genes known as degenenns that arc required for the function of touch-receptor neurons [50"°]. These genes share a considerable DNA sequence similarity to the amiloride-sensitive sodium channel. These data suggest, therefore, that dysfunction of neuronal ion channels in C. elegans can cause neurodegcneration. Another C. elegans mutant has also implicated ion channel mutation in neurodegeneration. The deg-3 gene encodes a nicotinic AChR ct subunit. A mutation in this gene causes degeneration of a small set of neurons in (7. elegans [51"]. Although the mechanism has not been tested specificall% a similar mutation in the chick protein produces channels that desensitize slowly. Additional evidence that channel hyperactivity is the mechanism for this phenomenon is indicated by the fact that a nicotinic receptor antagonist ameliorated the phenotype. Molecular genetic studies in mice have also implicated voltage-gated sodium channels in neurodegeneration. A mouse mutant known as 'motor endplate disease' [52"] is characterized by progressive paralysis, severe muscle atrophy, degeneration of Purkinje cells and death. A mutation in the voltage-gated sodium channel gene, SCNSA, has been implicated in this disorder [52°]; the
Pethophysiology of ion channel mutations Keating and Sanguinetti
331
mutation leads to a loss of SCN8A expression. As this is a recessive diso/der, these data indicate that a loss of SCN8A function contributes to neuronal degeneration.
delayed rectifier potassium current (IKr) in cardiac myocytes. This established HERG as the gene encoding cc subunits that form the IKr potassium channel. Because block of IKr is a known mechanism for drug-induced cardiac arrhythmias, the finding that HERG encodes IKr channels provided a mechanistic link between inherited and acquired cardiac arrhythmias.
Conclusions
5.
Trudeau M, Warmke J, Ganetzky B, Robertson G: HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 1995, 269:92-95.
6.
Spector P, Curran M, Keating M, Banguinetti M: Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Circ Res 1996, 78:499-603.
Mutations in voltage-gated potassium and chloride channels have been implicated in several muscle and neural disorders. These mutations are thought to cause abnormal cellular repolarization and increase cellular excitability. Depending on the organ involved, these inherited disorders can lead to cardiac arrhythmias, myotonia, or ataxia. In addition, it is now clear that dysfunction of ion channels can contribute to abnormal neural differentiation and neural degeneration. It seems likely that inherited and acquired dysfunction of ion channels will eventually prove important in a large number of disorders involving excitable and nonexcitablc cells.
Note added in proof Mutations in 6"LCNS, the gene encoding a chloride channel expressed in the kidney, cause three types of inherited hypercalciuric nephrolithiasis [53]. These mutations lead to the loss, or greatly diminish the function of, channels expressed in Xenopusoocytes. It is not known where in the kidney this channel is expressed or how mutations in the protein alter resorption of calcium, leading to the formation of kidney stones.
7 •
Sanguinetti MC, Curran ME, Spector P and Keating MT: Spectrum of HERG K+ channel dysfunction in an inherited cardiac arrhythmia. Proc Nat/Acad Sci USA 1996, 93:2208-2212. Here, we demonstrated that LQT-associated mutations in HERG cause loss, alteration and dominant-negative suppression of HERG function. Reduced repolarizing HERG potassium current presumably leads to increased myocellular excitability and arrhythmia susceptibility. 8. •.
Browne D, Gancher S, Nutt J, Brunt E, Smith E, Kramer P, Litt M: Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNAI. Nat Genet 1994, 8:136-140. This manuscript was the first to demonstrate that mutations in the human brain potassium channel gene, KCNA 1, cause episodic ataxia and myokymia (muscle rippling). 9.
10. •
Adelman J, Bond C, Pessia M, Maylie J: Episodic ataxia results from voltage-dependent potassium channels with altered functions. Neuron 1995, 15:1449-1454. This manuscript showed that mutations in the voltage-dependent delayed rectifier potassium channel gene, KCNA1, associated with episodic ataxia cause loss, alteration and dominant-negative suppression of channel funclion. 11.
George A, Crackower M, Abdalla J, Hudson A, Ebers G: Molecular basis of Thomsen's disease (autosornal dominant myotonia congenita). Nat Genet 1993, 3:305-310.
1 2.
Steinmeyer K, Lorenz C, Pusch M, Koch M, Jentsch T: Multimeric structure of CLC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen). EMBO J 1994, 13:737-743.
13.
Gronemeier M, Condie A, Prosser J, Steinmeyer K, Jentsch T, Jockusch H: Nonsense and missense mutations in the muscular chloride channel gene CLC-1 of myotonic mice. J Big~ Chem 1994, 269:5963-5967.
14.
Heine R, George A, Pika U, Deymeer F, Rudel R, Lehmann-Horn F: Proof of a non-functional muscle chloride channel in recessive myotonia congenita (Becker) by detection of a 4 base pair deletion. Hum Mo/Genet 1994, 3:1123-1128.
16.
Lorenz C, Meyer-Kleine C, Steinmeyer K, Koch M, Jentsch T: Genomic organization of the human muscle chloride channel CIC-1 and analysis of novel mutations leading to Becker-type myotonia. Hum Mol Genet 1994, 3:941-946.
16.
George A, Sloan-Brown K, Fenichel G, Mitchell G, Spiegel R, Pascuzzi R: Nonsense and missense mutations of the muscle chloride channel gene in patients with myotonia congenita. Hum Mo/ Genet 1994, 3:2071-2072.
Acknowledgements T h e authors thank H Katchuk,
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.
of special interest of outstanding interest Jiang C, Atkinson D, Towbin JA, Lehmann M, Splawski I, Li H, Taggart RT, Timothy K, Schwartz PJ, Vincent GM, Moss AJ, Keating MT: Two long QT syndrome loci map to chromosomes 3 and 7 with evidence for further heterogeneity. Nat Genet 1994, 8:141-147.
2. ••
Curran ME, Splawski I, Timothy K, Vincent GM, Green E, Keating MT: A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Ceil 1995, 80:795-803. This manuscript demonstrated that mutations in HERG, a putative potassium channel gene, cause an inherited cardiac arrhythmia, LQT. The discovery was the first to demonstrate that mutations in cardiac ion channel genes cause susceptibility to life-threatening arrhythmias. 3. ••
Wang Q, Curran M, Splawski I, Burn T, Millholland J, VanRaay T, Shen J, Timothy K, Vincent GM, De Jager T et al.: Positional cloning of a novel cardiac channel gene: KVLQT1 mutations cause cardiac arrhythmias, Nat Genet 1996, 12:1-23. In this manuscript, positional cloning techniques were used to identify a novel cardiac potassium channel gene, KVLQT1, and demonstrate that mutations in this gene cause the most common form of inherited LOT syndrome. KVLQT1 is predicted to be a voltage-gated potassium channel important for myocellular repolarization. LQT-associated mutations were predicted to have a dominant-negative effect on KVLGT1 function. 4. =•
Sanguinetti MC, Jiang C, Curran ME, Keating MT: A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Ceil 1995, 81:299-307 In this manuscript, we demonstrated that the biophysical properties of HERG expressed in Xenopus oocytes were nearly identical to the rapidly activating
Browne D, Brunt E, Griggs R, Nutt J, Gancher S, Smith S, Litt M: Identification of two new KCNA1 mutations in episodic ataxia/myokymia families. Hum Mol Genet 1995, 4:1671-16?2.
17 •
Pusch M, Steinmeyer K, Koch M, Jentsch T: Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CLC-1 chloride channel. Neuron 1995, 15:1455-1463, This manuscript provides the first demonstration that mutations in the skeletal muscle chloride channel, CLC-1, cause a dramatic shift in the voltage sensitivity of expressed channels, leading to a loss of function. These mutant channels cannot contribute to repolarization of skeletal muscle action potential, leading to myotonia. 18. •.
Wang Q, Shen J, Splawski I, Atkinson DL, Li Z, Robinson J, Moss A, Towbin J, Keating MT: SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome, Ceil 1995, 80:805-811. This work was the first to demonstrate that mutations in the cardiac sodium channel gene SCN5A cause LQT syndrome. These findings suggested that the cellular mechanism of the disease results from altered channel inactivation properties and continued depolarizing sodium current late in the plateau phase of the cardiac action potential.
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Genetics of disease
19.
Wang Q, Shen J, Li Z, Timothy K, Vincent GM, Priori S, Schwartz P, Keating MT: Cardiac sodium channel mutations in patients with long QT syndrome, an inherited cardiac arrhythmia. Hum Mol Genet 1995, 4:1603-1607.
20. Bennett P, Yazawa K, Makita N, George A: Molecular mechanism • for an inherited cardiac srrhythmia. Nature 1995, 376:683-685. This manuscript was the first to demonstrate that LQT-associated mutations in the cardiac sodium channel gene cause destabilization of the channel's inactivation gate and late channel reopenings. 21.
Hoffman E, Lehmann-Horn F, Rudel R: Overexcited or inactive: Ion channels in muscle disease. Cell 1995, 80:681-686.
22.
Yang N, Zhou M, Ptacak L, Barchi R, Horn R, George F: Sodium channel mutations in paramyotonia congenita exhibit similar biophysical phenotypes in vitro. Proc Nat/Acad Sci USA 1994, 91:12785-12789.
23.
Zhou .I, Spier S, Beech J, Hoffman E: Pathophysiology of sodium channelopathles: con'elatlon of normal/mutant mRNA ratios with clinical phenotype in dominantly inherited periodic paralysis. Hum Mol Genet 1994, 3:1599-1603.
24. •-
Ptacek L, Tawil R, Griggs R, Engel A, Layzer R, Kwiecinski H, McManis P, Santiago L, Moore M, Fouad G e t aL: Dlhydropydrine receptor mutations cause hypokalemic periodic paralysis. Cell 1994, 77:863-868. This is the first demonstration that mutations in the skeletal muscle voltage gated calcium channel gene (also known as the dihydropydrine receptor gene) cause episodic weakness associated with low serum potassium levels- hypokalemic periodic paralysis. 25. ••
Jurkat-Rott K, Lehmann-Horn F, Elbaz A, Heine R, Gregg R, Hogan K, Powers P, Lapie P, Vale-Santos J, Weissenbach J, Fontaine B: A calcium channel mutation causing hypokalemlc periodic paralysis. Hum Mol Genet 1994, 3:141 5-141 g. This manuscript established that mutations in the dihydropydrine receptor gene cause hypokalemic periodic paralysis. 26.
Sipos I, Jurkatt-Rott K, Harasztosi Cs, Fontaine B, Kovacs L, Melzer W, Lehmann-Horn F: Skeletal muscle DHP receptor mutations alter calcium currents in human hypokalaemic periodic paralysis myotubes. J Physio11995, 483:299-306.
27. ••
Canessa C, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J-D, Rossier B: Amlloride-sensitlve epithelial Na+ channel is made of three homologous subunits. Nature 1994, 367:483-46?. This study defined the primary structure of the genes that encode the epithelial sodium channel, demonstrating that it comprises three homologous subunits that interact to form functional channels. 28.
Snyder P, McDonald F, Stokes J, Welsh M: Membrane topology of the smiloride-sensitive epithelial sodium channel. J Bio/ Chem 1994, 269:24379-24383.
29. • ~,
Shimkets R, Warnock D, Bositis C, Nelson-Williams C, Hansson J, Schambelan M, Gill J, Ulick S, Milora R, Finding Jet al.: Liddle's syndrome: heritable human hypertension caused by mutations in the I~ subunit of the epithelial sodium channel. Cell 1994, 79:407-414. Here, the authors demonstrate that mutations in the ~ subunit of the epithelial sodium channel cause Liddle syndrome (pseudo aldosteronism), an autosoreal dominant form of human hypertension. The data presented suggest that the mechanism of Liddle syndrome is constitutive activation of an amiloridesensitive distal nephronal epithelial sodium channel. These mutations lead to increased sodium resorption by the kidney and susceptibility to hypertension. 30. •
Hansson J, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton R: Hypertension caused by a truncated epithelial sodium channel 7 subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 1995, 11:76-82. This manuscript showed that mutations in the Y subunit of the epithelial sodium channel cause susceptibility to hypertension in Liddle syndrome. 31. •
Schild L, Caness C, Shimkets R, Gautschi I, Lifion R, Rossier B: A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Nat/Acad Sci USA 1995, 92:569g-5703. In this manuscript, the authors expressed Liddle syndrome associated mutant 13subunits of the epithelial sodium channel with wild-type ~ and y subunits in Xenopus oocyles, demonstrating an increase in the macroscopic amiloride-sensitive sodium current. 32. •
Snyder P, Price M, McDonald F, Adams C, Volk K, Zeiher B, Stokes J, Welsh M: Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel. Cell 1995, 83:969-978.
This is the first demonstration that Liddle syndrome associated mutations in the epithelial sodium channel genes cause an increase in the number of sodium channels in the apical membrane and do not alter single-channel conductance or open-state probability. 33.
Jung J, Preston G, Smith B, Guggino W, Agre P: Molecular structure of the water channel through aquaporin CHIP. J Biol Chem 1994, 269:14648-14654.
34. •-
Deen P, Verdijk M, Knoers N, Wieringa B, Monnens L, Van Os C, Van Oost B: Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science 1994, 264:92-95. This was the first demonstration that the aquaporin-2 water channel was required for concentration of urine by the kidney. These findings suggested that mutations in the aquaporin-2 water channet gene might cause diabetes insipidus. Support for this hypothesis was demonstrated in one patient. 35. •
Van Lieburg A, Verdijk M, Knoers V, Van Essen A, Poresmans W, Mallmann R, Monnens L, Van Oost B, Van Os C, Deen P: Patients with autosomal nephrogenic diabetes insipidus homozygous for mutations in the aquaporin 2 water-channel gene. Am J Hum Genet 1994, 55:648-652. This manuscript identified missense mutations and a single nucleotide deletion in the aquaporin 2 gene in three individuals with nephrogenic diabetes insipidus. Expression of the mutants in Xenopus oocytes showed that the resultant channels were nonfunctional. These data indicate that mutations in the aquaporin-2 water channel gene can cause autosomal recessive nephrogenic diabetes insipidus. 36. •
Deen P, Croes H, Van Aubel R, Ginsel L, Van Os C: Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes instpidus are impaired in their cellular routing. J Ch'n Invest 1995, 95:2291-2296. This study presented immunolocalization data suggesting that diseaseassociated mutant forms of the aquaporin-2 water channel proteins fail to reach the plasma membrane, leading to nonfunctional water channels. 37.
Preston G, Smith B, Zeidel M, Moulds J, Agre P: Mutations in aquaporin-1 in phenofypically normal humans without functional CHIP water channels. Science 1994, 265:1585-1587.
38. •
Gunderson K, Kopito R: Conformational states of CFTR associated with channel gating: the role of ATP binding and hydrolysis. Cell 1995, 82:231-239. In this manuscript, electrophysiologic analyses of wild-type and mutant CFTR channels indicated that binding of ATP causes a reversible transition from the closed to an open state. Transition from this open state requires ATP hydrolysis. 39. •
Schwiebert E, Egan M, Hwang T, Fulmer S, Allen S, Cutting G, Guggino W: CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATR Ceil 1995, 81:1063-1073. Physiological studies have suggested that CFTR can interact to modulate outwardly rectifying chloride channels in addition to being a chloride channel itself. Data provided in this manuscript indicated that CFTR regulates outwardly rectifying chloride channels by triggering the transport and release of ATP. ATP, in turn, activates non-CFTR chloride channels. 40.
Reisin I, Prat A, Abraham E, Amara J, Gregory R, Ausiello D, Cantiello H: The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J Biol Chem 1994, 269:20584-20591.
41. •
Jensen 1", Loo M, Pind S, Williams D, Goldberg A, Riordan J: Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 1995, 83:129-135. CFTR levels are tightly regulated, in part by protein degradation. Proper trafficking in early degradation of mutant CFTR proteins is thought to be mechanistically important in cystic fibrosis. This manuscript demonstrated that the molecular mechanisms of CFTR processing include degradation in the proteasome. 42. Ward C, Omura S, Kopito R: Degradation of CFTR by the • ubiquitin-proteasome pathway. Cell 1995, 83:121-127. This manuscript demonstrated that the ubiquitin-proteasome pathway is important for CFTR degradation. 43. •
Sine S, Ohno K, Bouzat C, Auerbach A, Milone M, Pruitt J, Engel A: Mutation of the acetylcholine receptor ~ subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity. Neuron 1995, 15:229-239. This study demonstrated that mutations in the AChR ct subunit cause slowchannel myasthenic syndrome in several individuals. Electrophysiological analysis revealed a decreased rate of acetylcholine disassociation, causing the mutant receptors to open repeatedly during ligand occupancy, thereby altering the synaptic response. 44.
Ohno K, Hutchinson D, Milone M, Brengman J, Bouzat C, Sine S, Engel A: Congenital myasthenic syndrome caused by
P a t h o p h y s i o l o g y o f ion channel mutations Keating and Sanguinetti
prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the E subuniL Proc Nat/Acad Sci USA 1995, 92:758-762. 45.
Gomez C, Gammack J: A leucine-to-phenylalanine substitution in the acetylcholine receptor ion channel in a family with the slow-channel syndrome. Neuro/ogy 1995, 45:982-985.
46. •
Steinlein O, Mulley J, Propping P, Wallace R, Phillips H, Sutherland G, Scheffer I, Berkovic S: A missense mutation in the neuronal nicotinic acetylcholine receptor c¢4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 1995, 11:201-203. This study presented data suggesting that a missense mutation in the neuronal nicotinic AChR may be the cause of autosomal dominant nocturnal frontal lobe epilepsy in humans. The functional consequences of this mutant are not yet known. 47.
Shiang R, Ryan S, Zhu Y-Z, Hahn A, O'Connell P, Wasmuth J: Mutations in the c~1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nat Genet 1993, 5:351-358.
48. •
Rajendra S, Lynch J, Pierce K, French C, Barry P, Schofield P: Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor. J Bio/Chem 1994, 269:18739-18742. This manuscript demonstrated that mutations in the glycine receptor, a ligand-gated chloride channel associated with Startle disease, disrupt receptor function by causing a dramatic decrease in sensitivity to glycine. Startle disease mutations in the glycine receptor reduce the efficiency of glycinergic inhibitory neurotransmission. 49. •.
Patil N, Cox D, Bhat E), Faham M, Myers R, Peterson A: A potassium channel mutation in weaver mice implicates
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membrane excitability in granule cell differentiation. Nat Genet 1995, 11:126-129. This manuscript demonstrated that a missense mutation in a G-protein-coupled inward rectifier potassium channel gene cause the weaver phenotype in mice. Homozygous animals suffer from severe ataxia that is thought to result from a block in neuronal differentiation in the cerebellum. 50. •o
Huang M, Chalfie M: Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans. Nature 1994, 367:467-470. This study characterizes two genes in C. elegans that are critical for mechanosensory transduction, and, from DNA sequence homology, are similar to the amiloride-sensitive sodium channel c~, 13and ~, subunits. 51. •
Treinin M, Chalfie M: A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans. Neuron 1995, 14:871-877. This manuscript demonstrated that a gain-of-function mutation in an AChR subunit leads to degeneration of a set of neurons in C. elegans. Because antagonists of nicotinic AChRs suppress the mutant phenotype, these data suggest that channel hyperactivity may underlie neurodegeneration in this model. 52. •
Burgess D, Kohrman D, Gait J, Plummer N, Jones J, Spear B, Meisler M: Mutation of a new sodium channel gene, ScnSe, in the mouse mutant 'motor endplate disease'. Nat Genet 1995, 10:461-465. This manuscript demonstrated that mutation of a brain sodium channel gene is responsible for neurodegeneration in the mouse mutant known as 'motor endplate disease'. 53.
Lloyd SE, Pearce SHS, Fisher SE, Steinmeyer K, Schwappach B, Scheinman S.I, Harding B, Brolino A, Devoto M, Goodyer Pet al: A common molecular basis for three inherited kidney stone diseases. Nature 1996, 379:445-449.