Channelopathies: Episodic Disorders of the Nervous System
2001; Wiley; Volume: 42; Issue: s5 Linguagem: Inglês
10.1111/j.1528-1167.2001.0s007.x
ISSN1528-1167
Autores Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoThe field of channelopathies is a newly recognized group of disorders named for the site of the molecular defects-voltage- and ligand-gated ion channels. Although voltage-gated ion channel mutants have been recognized for some time in organisms such as Drosophila, the first channelopathy in humans was first reported within the last decade 1. The recognition of this group of disorders began with definition of the molecular basis of a group of unusual muscle disorders called the periodic paralyses and nondystrophic myotonias 2. Interestingly, this group of muscle disorders shares some interesting phenotypic features with a number of seemingly disparate human diseases that involve not only skeletal muscle, but also brain and heart 3, 4. Some similarities that exist among these different disorders include their episodic nature, similarities with regard to factors that precipitate attacks, therapeutic agents that can help to treat or prevent attacks, and in some cases, a degenerative component that arises in addition to the episodic attacks. The study of these diseases, along with the recognition of common clinical and pathophysiologic themes among these disorders, has led to tremendous growth in our understanding of these diseases and the hope of developing better therapies. Voltage-gated ion channels are proteins critical for establishment of the resting membrane potential in muscle and the ability of these membranes to generate action potentials. Voltage-gated opening of sodium channels results in the genesis of "all-or-none" action potentials. These channels close over the course of a few milliseconds. A simultaneous effect of depolarization of the membrane (albeit on a slower time scale) is the opening of voltage-gated potassium channels. Along with the inactivation of the voltage-gated sodium channels, the movement of positive potassium ions out of the cell through voltage-gated potassium channels leads to a relatively rapid repolarization of the muscle membranes. Chloride channels are responsible for a majority of the polarity of resting membranes. The voltage-gated L-type calcium channel in skeletal muscle, also known as the dihydropyridine receptor because dihydropyridines block this channel, allows conductance of calcium into the cell. It is likely that this calcium is important through signaling pathways to effect other downstream changes of the muscle membrane, but the calcium conductance itself is not important directly to the depolarization of the muscle membrane. Interestingly, this voltage-gated calcium channel is known to serve as a voltage sensor for excitation–contraction coupling. Depolarization of the muscle membrane leads to changes in the calcium channel protein, which in turn interacts with the ryanodine receptor to open slow-release calcium channels in the sarcoplasmic reticulum (SR). It is these slow-release channels and the SR calcium stores that lead to elevations in cytosolic calcium that result in the ultimate contraction of muscle. Potassium channels are membrane-bound tetrameric protein complexes. Each subunit is a polypeptide with six putative transmembrane segments (S1-6) (Fig. 1). Four identical subunits may associate to form a homomeric K channel. Alternatively, different subunits may assemble to form a heteromeric K channel. Mutagenesis studies have identified several critical functional domains in K channel. The S4 segments with positively charged arginine and lysine residues at every third position appear important in voltage sensing. The P region linking putative membrane-spanning S5 and S6 contains the K channel signature sequence that is highly conserved and critical for K selectivity of the channel pore. Diagram of a voltage-dependent potassium channel subunit, with an intracellular N-terminus, six transmembrane segments, and an intracellular C-terminus. The S4 segments with positively charged arginine and lysine residues at every third position are important in voltage sensing. Putative membrane-spanning S5 and S6 and the P region linking the two segments line the channel pore. Four subunits come together to form a potassium channel. The voltage-dependent sodium and calcium channels are a large group of homologous genes that also are homologous to the voltage-gated potassium channel genes. Unlike the potassium channel proteins in which four subunits must come together to form a homo- or heterotetrameric functional channel, sodium and calcium channel subunits have evolved to include in a single transcript four domains (I-IV) in tandem, each with six transmembrane segments (S1-6) homologous to voltage-gated potassium channel genes (Fig. 2). It is thought that this phenomenon resulted from the duplication of a progenitor "potassium-like" channel that duplicated and then reduplicated in the genome. The large, pore-forming α subunit alone is sufficient for cation permeability, pharmacologic specificity, gating, and voltage sensitivity. The auxiliary subunits modulate channel biophysical properties and biosynthesis. Voltage-dependent sodium channels are composed of a pore-forming, voltage-sensing α subunit and a transmembrane β subunit. Voltage-dependent calcium channels are heteromeric complexes composed of a pore-forming α1 subunit, a disulfide-linked membrane-anchored extracellular α2-δ subunit, an intracellular β subunit, and in muscle, a γ subunit. Diagram of a subunit of a sodium channel or a1 subunit of a calcium channel, with four homologous domains, each with six transmembrane segments like those described in Fig. 1. Voltage-gated chloride channels have been discovered more recently, and much less is known about their structure. These proteins have 13 hydrophobic segments, but more recent biochemical and immunologic data suggest that not all of these traverse the membrane. It appears that voltage-gated chloride channels form multimeric functional units, but the exact stoichiometry of these channels is not entirely clear. There are no recognized auxiliary subunits of the voltage-gated chloride channels at this time. Periodic paralyses and nondystrophic myotonias include a number of distinct clinical entities as well as some intermediate forms of the various disorders. Myotonia congenita is a group of muscle disorders named for the prominent muscle hyperexcitability or myotonia that is seen in these patients. This myotonia is classic myotonia with the phenomenon of "warm up." These patients experience extreme muscle stiffness due to delayed relaxation from repetitive electrical activities in muscle, but this myotonia subsides as their muscles warm up with use. Onset of symptoms is generally in childhood through early adult life. These patients often have hypertrophy of their muscles and a Herculean appearance as a result of their myotonia. Two distinct forms of myotonia congenita are recognized. The first, named for Julius Thompson who described the disease, is an autosomal dominant form of myotonia congenita 5. These patients do not have degeneration of their muscles even after years of having the disease. An autosomal recessive form of myotonia congenita was described by Becker 6. These patients have myotonia with warm-up phenomenon, may have transient bouts of weakness after periods of disuse, and sometimes myopathy develops as part of the disease. Hyperkalemic periodic paralysis is a disorder with myotonia like that seen in the previously described disorders. These patients can also have a transition of the muscle membrane hyperexcitability to inexcitability in the form of episodic weakness. This weakness may be so dense as to cause a transient flaccid quadriparesis. However, the disorder does not affect the diaphragm, and patients are therefore able to continue breathing. First described in 1951 by Tyler 7, this disorder is named because of the ability to precipitate attacks in patients by administrating a sufficient dose of oral potassium. During spontaneous attacks, patients may have elevated potassium levels, although potassium is frequently in the normal range. Attacks of weakness can be precipitated by foods high in potassium, rest after vigorous exercise, and stress and fatigue. The disease is transmitted as an autosomal dominant trait, although sporadic cases are sometimes encountered. Percussion and action myotonia are frequently elicited clinically, and prominent myotonia can be noted on a electromyographic examination. Patients benefit dramatically from treatment with carbonic anhydrase inhibitors. Paramyotonia congenita is yet another disorder in which myotonia is present, although the myotonia is somewhat different, as it does not show the classic warm-up phenomenon, but rather is paradoxic. That is, patients frequently have worsening of the myotonia with repeated muscle action. This can be most prominently seen in the orbicularis oris muscles when patients forcefully close their eyes repeatedly and on such a maneuver, the myotonia of these muscles becomes increasingly severe to the point that patients might have difficulty opening their eyes altogether. Of interest, this disorder is a temperature-sensitive mutant of humans. With cooling of their muscles, these patients have worsening of their myotonia and then transition of the hyperexcitability into paralysis. This can be measured quantitatively using electrodiagnostic maneuvers 8. Paramyotonia congenita is transmitted as an autosomal dominant trait, and like hyperkalemic periodic paralysis, these patients have worsening of their symptoms with stress, fatigue, and rest after vigorous exercise. Patients may be hypo-, normo-, or hyperkalemic during attacks. The classic paramyotonia congenita patients are generally hypokalemic during attacks; those with hyperkalemia seem to represent a clinical entity somewhere in the spectrum between classic paramyotonia congenita and hyperkalemic periodic paralysis. Like the hyperkalemic periodic paralysis patients, these patients benefit dramatically from treatment with carbonic anhydrase inhibitors. Potassium-activated myotonia is a disorder in which patients clinically appear to have myotonia congenita, but their myotonia fluctuates, worsens when potassium is administered, and improves with carbonic anhydrase inhibitors 9. Interestingly, these patients do not develop attacks of weakness. This disorder is transmitted as an autosomal dominant trait. Hypokalemic periodic paralysis is a disorder of episodic weakness in which myotonia is not seen. These patients are generally hypokalemic during an attack, and attacks can be precipitated by lowering potassium levels with administration of glucose and insulin. Furthermore, these patients' attacks can be precipitated by stress, fatigue, and rest after vigorous exercise. Dietary precipitants include high-carbohydrate meals and salt load. Hyperkalemic periodic paralysis is transmitted as an autosomal dominant trait, although frequent sporadic cases are seen. Patients' potassium levels during hypokalemia generally remain >2 mM. Potassium levels <2 mM during an attack of weakness in a sporadic case raises the possibility of thyrotoxic hypokalemic periodic paralysis, a nonmendelian form of the disorder, which is seen only during periods of thyrotoxicosis 2. The episodic weakness in patients with hyperkalemic periodic paralysis, paramyotonia congenita, and hypokalemic periodic paralysis benefit from treatment with the carbonic anhydrase inhibitors acetazolamide (Diamox) and daranide. Linkage analysis in large pedigrees with hyperkalemic periodic paralysis established that the gene for this disorder resided on chromosome 17q 10, 11. Mapping data showed that the paramyotonia congenita and the potassium-activated myotonia phenotypes also map to the same locus 12, 13. Subsequently, a sodium channel gene in this region of chromosome 17q was cloned and characterized and shown to be the site of mutations in hyperkalemic periodic paralysis 1, 14, paramyotonia congenita 15, 16, and potassium-aggravated myotonia 17. The data supporting that this sodium channel gene, SCN4A, was the disease-causing gene included the following: (a) Mutations segregated with the phenotype; (b) They involved highly conserved amino acid residues; (c) These mutations were not found in control individuals; and (d) Some of them occurred as de novo mutations in patients with sporadic disease. A large number of sodium channel mutations have been identified in patients with hyperkalemic periodic paralysis, paramyotonia congenita, and potassium-aggravated myotonia. Similar molecular approaches led to mapping of the hypokalemic periodic paralysis locus to chromosome 1q 18, 19. Subsequently, a voltage-gated calcium channel at this chromosome 1 locus was cloned. This calcium channel gene encodes an L-type calcium channel, and three patient-specific mutations have been identified 18. The mutations occur at highly conserved residues in the S4 segments of either domain 2 and 4. The domain 4 mutations occur at analogous position to the most common paramyotonia congenita mutations in the homologous SCN4A sodium channel 18. Genetic linkage analysis in families segregating alleles for both recessive and dominant forms of myotonia congenita showed that both of these phenotypes were linked to a locus on chromosome 7q 20, 21. Subsequent work has identified a voltage-gated chloride channel at this locus, CLCN1, to be the site of defects in myotonia congenita 20, 21. Subsequently, a long list of mutations in this channel gene have been shown to cause dominant and recessive forms of myotonia congenita (reviewed in 22). Interestingly, some of these mutations are recognized to cause both dominant and recessive forms 23. Although it is likely that this is a result of polymorphisms in the chloride channel gene itself that may modulate the effect of such mutations, no data are available to substantiate this hypothesis. Many of these chloride channel mutants have been expressed in vitro (either in oocytes or by transfection of mammalian cells growing in culture), and the physiologic abnormalities in myotonia congenita are being characterized. Discussion of the functional consequences is beyond the scope of this chapter, but they are leading to new insights into these disorders. Episodic ataxia is a rare inherited syndrome of intermittent ataxia of early onset with no known inborn errors of metabolism. There are two distinct forms, both with episodic attacks of ataxia responsive to acetazolamide (AZM), features reminiscent of periodic paralysis and suggestive of underlying ion channel abnormalities. Episodic ataxia type 1 (EA1), an autosomal dominant disorder involving both the central and the peripheral nervous system, is characterized by attacks of ataxia and persistent myokymia. Episodes of ataxia, with gait imbalance and slurring of speech, occur spontaneously or can be precipitated by sudden movement, excitement, or exercise. The attacks generally last from seconds to several minutes at a time and may recur many times a day. Myokymia, or muscle rippling resulting from motor nerve hyperexcitability, may be observed particularly around the eyes or in the small hand muscles. Subclinical rhythmic muscle activity may be demonstrated by electromyography. AZM, a carbonic anhydrase inhibitor, may be effective in preventing attacks of ataxia 24. The age at onset, frequency of attacks, and severity may vary widely among family members. Episodic ataxia type 2 (EA2) is an autosomal dominant disorder with episodes of markedly impaired truncal ataxia lasting hours to days, with interictal eye movement abnormalities. Exertion and stress commonly precipitate the episodes. Often the episodes of ataxia respond to AZM 25. In some individuals, there may be a gradual baseline ataxia with evidence of cerebellar atrophy. Affected patients also may have migraine; some even complain of basilar migraine 25. Linkage analysis of several large pedigrees with EA1 mapped the disease locus to 12p13, near a cluster of three K channel genes: KCNA1, KCNA5, and KCNA626. Based on the clinical phenotype and its analogy to episodic disorders of muscle, ion channel genes were considered good candidate genes for EA1. Browne et al. 24 serendipitously focused on KCNA1: the individual who led these investigators to exclude KCNA5 and KCNA6 later turned out to represent a phenocopy. KCNA1, which encodes Kv1.1, a delayed-rectifier K channel, has no intervening introns in its 1,488-bp sequence. Analysis of the single exon in KCNA1 identified point mutations in four unrelated EA1 pedigrees 24. These mutations fulfilled several criteria for disease-causing mutations, as they segregated with the phenotype, involve highly conserved amino acid residues in the potassium channel gene product, and were not found in controls. Additional mutations in the same gene were subsequently identified in other EA1 pedigrees (reviewed in 22). There is not an animal model for episodic ataxia type 1 with missense mutations in KCNA1. Smart et al. 27 generated Kv1.1 null mice that have frequent spontaneous seizures but display no evidence of ataxia. The disease locus in EA2 in several pedigrees was localized to chromosome 19p. Earlier, the disease locus of half of the pedigrees with familial hemiplegic migraine (FHM) was mapped to the same locus 28. FHM is a dominantly inherited subtype of migraine with aura, characterized by recurrent attacks of migrainous headache with ictal hemiparesis. Recovery is generally complete, with a normal interictal physical examination. The onset is often early in life. Overlap in symptoms between hemiplegic migraine and basilar migraine suggests that hemiplegic migraine may be a form of basilar migraine. A calcium channel subunit gene mapping to 19p was an ideal candidate gene for EA2 and FHM. Ophoff et al. 25 first defined the complex gene structure of CACNA1A, which spans 300,000 bp and consists of 47 exons that encode the α1A subunit with 2,261 amino acids. These investigators then analyzed the exons and flanking introns of CACNA1A and identified missense mutations in pedigrees affected with FHM and point mutations that result in premature stop or interfere with splicing in EA2 families. Subsequently, small expansions of a polymorphic CAG repeat in the same gene were identified in some ataxic patients with a condition since designated SCA6, a dominantly inherited progressive cerebellar ataxia clinically indistinguishable from other dominant ataxic syndromes 29. Some patients experience fluctuating symptoms, similar to episodic ataxia. Yue et al. 29a. identified a missense mutation in a family with severe progressive ataxia and superimposed episodes of vertigo and ataxia. Providing additional evidence supporting CACNA1A as the disease-causing gene in episodic ataxia, these researchers identified. in a patient with episodic ataxia but no family history, a de novo mutation that predicts a premature stop codon. Of note, mutations in genes encoding calcium channel subunits have been identified in a number of recessive mouse mutants with ataxia (reviewed in 22). Homozygous point mutation in the α1A gene (P1802L, Domain II P-region) causes epilepsy and ataxia in mutant mouse tottering tg22. Novel sequences in the intracellular COOH-terminus of the α1A subunit also resulted in a mutant mouse phenotype, leaner (tgla), with ataxia and epilepsy. Burgess et al. 29b. reported that mutation of a calcium channel gene with a predicted deletion of the highly conserved α1-binding motif in the β4 subunit was associated with seizures and ataxia in a mutant mouse, lethargic lh. Characterization of the genetic defects in the stargazer and waggler mutants lead to the identification of a neuronal calcium channel γ subunit. Congenital myasthenic syndromes are a group of rare, hereditary, non-immune-mediated disorders of neuromuscular transmission. Depolarization of the motor neuron activates presynaptic voltage-gated calcium channels to allow calcium ion influx to trigger the release of vesicles containing acetylcholine (ACh). ACh released into the synapse binds to nicotinic ACh receptors (nAChRs) clustered in end plate (EP) on the postsynaptic muscle surface membrane. The entry of cations through the activated nAChRs leads to depolarization (end-plate potential), thus activating voltage-gated sodium channels and ultimately muscle contraction. ACh is cleared from the synaptic cleft by acetylcholinesterase (AChE). Defects involving the presynaptic, synaptic, and postsynaptic components of the neuromuscular junction have been identified in different pedigrees affected with myasthenic symptoms. Postsynaptic dysfunction accounts for the majority of congenital myasthenic syndromes. A wealth of detailed studies of the physiologic, pharmacologic, and molecular properties of nAChR greatly facilitated the clinical characterization and subsequent identification of nAChR dysfunction causing congenital myasthenia. In particular, in two pedigrees with congenital myasthenic syndrome, Engel et al. 30 performed electrophysiologic studies to demonstrate markedly prolonged end-plate potentials, which led to the hypothesis that mutations in one or more nAChR channel subunits could underlie the abnormal kinetics observed. This hypothesis was subsequently confirmed by the identification of specific mutations in genes encoding different subunits of the nAChR in slow-channel congenital myasthenic syndrome (SCCMS). In addition, other mutations in nAChR subunit genes were associated with fast channel syndrome and nAChR deficiency. The nicotinic acetylcholine receptor (nAChR) complexes are ligand-gated channels expressed in skeletal muscles and brain. Five different subunits exist: α1-9, β1-4, γ, δ, ε. These subunits are encoded by different genes on different chromosomes. They are homologous, each with a large N-terminal extracellular domain, four transmembrane regions, with a large intracellular loop between M3 and M4 (Fig. 3) Diagram of a subunit of nicotinic acetylcholine receptor channel or inhibitory glycine receptor channel with four putative transmembrane domains. The subunit composition at the neuromuscular junction is fixed: (α1)2(β1)δγ in the fetal form and (α1)2(β1)δε in the adult form. M2 from each subunit lines the channel pore. A leucine residue at homologous locations in each M2 segment forms a hydrophobic ring critical for channel gating. There are two binding sites for ACh, located at the interface between N-terminal hydrophilic domains of α-δ and α-ε or α-γ. The snake neurotoxin α-bungarotoxin irreversibly blocks these binding sites and has been useful in labeling and counting nAChRs. Binding of two ACh molecules induces conformational changes of the channel complex to an open state to allow mostly sodium but also calcium to enter the cell. The channel may undergo further voltage-dependent conformational changes before it closes and ACh dissociates from the receptor channel complex. Slow-channel congenital myasthenic syndrome (SCCMS) is an autosomal dominant hereditary condition characterized clinically by weakness, fatigability, and progressive muscle atrophy 30. Patients generally are seen early, with poor head control and weak suck during infancy. Weakness of skeletal muscles, particularly bulbar, cervical, and hand-extensor muscles, may be present at birth. The age at onset and severity may vary among family members. Laboratory testing reveals no presence of anti-AChR antibodies. Morphologic studies at the light-microscopic level show focal end-plate myopathy. Ultrastructural studies reveal degenerative changes in the basement membrane, postsynaptic membrane, and the synaptic organelles of the neuromuscular junction. Electrophysiologic testing demonstrates many abnormalities. At a normal neuromuscular junction, a single stimulus should elicit a single compound muscle action potential (CMAP). In contrast, a single stimulus in SCCMS may elicit multiple potentials with decremental response, in addition to the initial CMAP. On repetitive stimulation of motor nerves, there is decremental electromyographic response. Intracellular recordings of muscle biopsies show prolonged decay of end-plate potentials. Single channel recordings demonstrate increased channel opening compared with control. Deficiency of nAChR is an autosomal recessive disorder causing congenital myasthenic syndrome with generalized weakness of early onset. The most striking abnormality in laboratory studies is the marked reduction in nAhR in motor end plate measured by α-bungarotoxin labeling. The patients usually respond favorably to AChE inhibitors. Immunocytochemical studies using labeled antibodies directed against the γ subunit have successfully demonstrated the presence of the immature subunit at the neuromuscular junction. There may be increased numbers of small end plates distributed over an increased span of muscle fiber surface. Electrophysiologic studies showed evidence of AChR with small conductance and slow kinetics characteristic of γ-containing rather than ε-containing channel complex. The low-affinity fast-channel syndrome is a rare, recessive condition in which patients generally have moderately severe myasthenic symptoms since birth 31, 32. Electrophysiologic studies revealed decremental CMAP and very small MEPPs. Single channel recordings from muscle biopsies of end plate showed infrequent, abnormally brief openings in response to ACh. Morphologic studies demonstrated normal end-plate structure, with normal numbers of AChRs and no evidence of myopathy. The abnormal "slow channel" kinetics suggests abnormal channels, which could result from mutations in one or more subunits of the nAChR. Patients with electrophysiologically confirmed slow-channel phenotype were screened for mutations in genes encoding the α1, β1, δ, ε subunits. Single-strand conformation polymorphism analysis of polymerase chain reaction (PCR)-amplified exons and flanking introns of all subunits of nAChR was performed. Direct sequencing of aberrant bands successfully identified mutations in different subunit genes. Several groups have reported recessive, heteroallelic mutations in the ε subunit gene in patients with nAChR deficiency or the low-affinity fast channel syndrome. Hereditary hyperekplexia, or startle disease, is a rare, highly penetrant autosomal dominant disorder characterized by an exaggerated startle response and hypertonia. The normal startle reflex is a primitive reflex with a complex, stereotyped pattern of motor behavior in response to unexpected sensory stimuli. The motor response generally consists of blinking, grimacing, flexion of neck and arms, and delayed abduction of the hand muscles. Patients with startle syndromes due to various neuropathologic conditions may have abnormally exquisite sensitivity or abnormally violent motor response to sudden stimuli. In particular, studying hereditary hyperekplexia has helped elucidate the pathophysiologic and genetic basis of abnormal startle response. A small kindred with "emotionally precipitated drop seizures," described by Kirstein and Silvferskiold 33 in 1958, was perhaps the first case report of hereditary hyperekplexia. Suhren et al. 34 subsequently described a major form and a minor form of excessive response to sudden stimuli in a large Dutch pedigree spanning five generations. The symptoms that characterize the major form consisted of transient hypertonia during infancy, an exaggerated startle response with generalized stiffening causing falls but no loss of consciousness, repetitive limb jerking particularly at night, hyperreflexia, and hesitant gait. Excessive startle without any associated symptoms characterizes the minor form. Symptoms of the major form of hereditary hyperekplexia could be seen early, as in two unrelated patients who had unusual fetal movements during gestation, with sudden forceful jerking lasting from seconds to minutes that increased in severity in response to external stimuli 35. Shortly after birth, both exhibited rigidity, nocturnal limb jerking, and an exaggerated startle response, which are typical findings in the neonatal period in patients with hereditary hyperekplexia. Unexpected stimuli, such as noises or normal handling, could precipitate massive, generalized spasms of skeletal muscles to cause apnea, cyanosis, even death, during infancy 36. Often mistaken for spastic quadriplegia, neonatal stiffness improves in early childhood 37. Delay in motor development is common because any sudden sound, touch, or movement could cause patients to stiffen and fall. Diffuse stiffness renders the patients completely powerless in breaking their falls. There is no loss of consciousness. Patients may develop an awkward slow wide-based gait because of fear of falling. Inguinal and abdominal hernia as well as generalized seizures independent of startle in patients with hyperekplexia also have been reported 34, 36. Anxiety, stress, fear, sleep deprivation, and menstruation exacerbate the symptoms. Physical examination usually reveals diffusely increased muscle tone during infancy that normalizes with age. Tapping of the forehead results in excessive head retraction. Deep tendon reflexes are generally hyperactive. The preservation of consciousness and the absence of epileptiform discharges on EEG distinguish hyperekplexia from startle epilepsy 37. Analysis of the electromyographic studies as well as evoked responses demonstrated the same startle pattern in control subjects as in patients with hyperekplexia, regardless of etiology 38, 39. The acoustic startle pathway is not completely understood but is thought to involve mainly the caudal brainstem. Lesion and stimulation studies identified a system, with signals traveling from the auditory nerve to the ventral cochlear nucleus, nuclei of the lateral lemniscus, nucleus reticularis pontis caudalis, reticulospinal tract, spinal interneurons, and finally, the lower motor neurons to innervate skeletal muscles. Lesions involving the startle circuit spanning the caudal brainstem and the spinal cord could lead to decreased startle. Excessive startle would indicate increased excitatory or decreased inhibitory input to the startle circuit. Linkage studies in several unrelated kindreds with autosomal dominant inheritance of hereditary hyperekplexia localized the disease gene to the distal portion of the long arm of chromosome 5 41. This region contained genes encoding subunits of γ-aminobutyric acid (GABA) receptor, glycine receptor, adrenergic receptor, and glutamate receptor. Radiation hybrid mapping analysis demonstrated that only the gene encoding the α1 subunit of the inhibitory glycine receptor (GLRA1) was within the disease gene region 42. GLRA1 was a good candidate gene for hyperekplexia because glycinergic transmission mediates recurrent inhibition of pontomedullary reticular neurons as well as spinal motor neurons. Screening by denaturing gradient gel electrophoresis combined with direct sequencing uncovered two different missense mutations in exon 6 of GLRA1 in affected members from four families, substituting an uncharged amino acid (leucine or glutamine) for arginine, a charged residue. Additional mutations in GLRA1 were subsequently identified in other hereditary and sporadic cases of hyperekplexia. Mice are excellent animal models for studying hyperekplexia in humans. When given sublethal doses of strychnine, which is a competitive glycine receptor antagonist, normal mice display hypertonia and an exaggerated startle reflex, reminiscent of the cardinal features of hyperekplexia in human. Furthermore, two autosomal recessive mouse mutants, spastic (spa) and spasmodic (spd), exhibit identical phenotypes with striking similarities to hyperekplexia in humans. Phenotypically normal at birth, the affected animals develop rigidity and fall in response to sudden tactile or acoustic stimuli after the second postnatal week. A lethal mutant, oscillator (ot), displays more severe symptoms and may be allelic to spd. The phenotypic similarities between spd and hyperekplexia suggest that mutations in glycine receptor gene may be responsible for the spd phenotype. The disease locus of spd mapped to a small region on mouse chromosome 11 that was homologous to the hyperekplexia-containing region on human chromosome 5q 43, 44. Direct sequencing showed that spd indeed was the mouse homologue of hyperekplexia and a specific missense mutation (G534T) was identified in spd, which would result in an Ala52Ser substitution in the mouse glycine receptor α1 subunit, sharing 99% peptide sequence homology with the adult human orthologue 45. Of note, the expression of glycine receptor isoforms is developmentally regulated, switching from the neonatal to the adult isoform around the second postnatal week in mice. The neonatal isoform of the glycine receptor contains α2 subunits only, whereas the adult isoform appears to be composed of three α1 subunits and two β subunits. That spd mice are phenotypically normal until after the second postnatal week is consistent with the expression of the mutation-containing α1 subunit gene of the adult isoform of glycine receptor. A growing list of paroxysmal neurologic disorders has been shown to result from mutations in ion channels (Table 1). The identification of specific mutations has made diagnosis and patient classification possible. Physiologic characterization of mutant channels will continue to reveal new functional domains. The finding that mutations in the auxiliary subunits may have the same clinical manifestations as mutations in the main, pore-forming channel subunits indicates that defects in any component (upstream or downstream) with which the channel protein interacts can lead to dysfunction. That mutations in distinct proteins may produce similar phenotypes provides insight to the functional role of the different channel proteins that must be carefully coordinated to carry out proper neurologic activities. In the case of congenital myasthenic syndromes, defects involving the presynaptic, synaptic, or postsynaptic component of the neuromuscular junction may interfere with normal neuromuscular transmission. It is intriguing that different mutations in the same gene produce different phenotypes (although with some overlap), as illustrated by the allelic disorders involving mutations in SCN4A and CACNA1A. Phenotypic variability among individuals within the same pedigree or different pedigrees with the same mutation suggests that other genetic and/or environmental factors must contribute to the phenotypic expression. Understanding the phenotypic expression of the rare, monogenic channelopathies presented in this chapter may help elucidate similar mechanisms in other paroxysmal neurologic disorders, such as familial paroxysmal dyskinesia, migraine, and epilepsy. Familial paroxysmal dyskinesia is a rare group of disorders characterized by episodic involuntary hyperkinetic movement 46. A familial paroxysmal choreoathetosis syndrome associated with progressive spasticity mapped to chromosome 1p 47, whereas paroxysmal kinesigenic dyskinesia not associated with spasticity mapped to chromosome 2q34 48. Ion channels are candidate genes for these episodic movement disorders. Because migraine shares many features with known channelopathies, ion channels are likely sites of genetic defects in this heterogeneous and possibly polygenic disorder. In addition to CACNA1A mutations discussed in this chapter, two other loci on chromosome 1q have been identified in other pedigrees with familial hemiplegic migraine, which is a rare form of migraine with aura 49, 50. Epilepsy also shares many features with known channelopathies; indeed, mutations have been identified in both ligand-gated and voltage-gated ion channel genes in different pedigrees with epilepsy 51-55. With the Human Genome Project and the development of DNA chip technology well under way, soon we will be able to easily identify genes and mutations. One of the challenges that we face is to understand how these ion channel mutations are translated into paroxysmal neurologic disturbances as well as fixed neurologic deficits. Studying the biophysical properties of individual ion channels is a good start. Other processes such as gene expression, alternative splicing, assembly, subcellular localization, modulation by protein kinases/phosphatases, interaction with synaptic machinery and cytoskeletal elements, and excitotoxicity are incompletely understood but clearly important in elucidating the underlying disease-causing mechanism and developing rational treatment. Acknowledgment: This study was funded by NIH.
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