The non-dystrophic myotonias: molecular pathogenesis, diagnosis and treatment.

The non-dystrophic myotonias are an important group of skeletal muscle channelopathies electrophysiologically characterized by altered membrane excitability. Many distinct clinical phenotypes are now recognized and range in severity from severe neonatal myotonia with respiratory compromise through to milder late-onset myotonic muscle stiffness. Specific genetic mutations in the major skeletal muscle voltage gated chloride channel gene and in the voltage gated sodium channel gene are causative in most patients. Recent work has allowed more precise correlations between the genotype and the electrophysiological and clinical phenotype. The majority of patients with myotonia have either a primary or secondary loss of membrane chloride conductance predicted to result in reduction of the resting membrane potential. Causative mutations in the sodium channel gene result in an abnormal gain of sodium channel function that may show marked temperature dependence. Despite significant advances in the clinical, genetic and molecular pathophysiological understanding of these disorders, which we review here, there are important unresolved issues we address: (i) recent work suggests that specialized clinical neurophysiology can identify channel specific patterns and aid genetic diagnosis in many cases however, it is not yet clear if such techniques can be refined to predict the causative gene in all cases or even predict the precise genotype; (ii) although clinical experience indicates these patients can have significant progressive morbidity, the detailed natural history and determinants of morbidity have not been specifically studied in a prospective fashion; (iii) some patients develop myopathy, but its frequency, severity and possible response to treatment remains undetermined, furthermore, the pathophysiogical link between ion channel dysfunction and muscle degeneration is unknown; (iv) there is currently insufficient clinical trial evidence to recommend a standard treatment. Limited data suggest that sodium channel blocking agents have some efficacy. However, establishing the effectiveness of a therapy requires completion of multi-centre randomized controlled trials employing accurate outcome measures including reliable quantitation of myotonia. More specific pharmacological approaches are required and could include those which might preferentially reduce persistent muscle sodium currents or enhance the conductance of mutant chloride channels. Alternative strategies may be directed at preventing premature mutant channel degradation or correcting the mis-targeting of the mutant channels.

The primary periodic paralyses: diagnosis, pathogenesis and treatment.

Periodic paralyses (PPs) are rare inherited channelopathies that manifest as abnormal, often potassium (K)-sensitive, muscle membrane excitability leading to episodic flaccid paralysis. Hypokalaemic (HypoPP) and hyperkalaemic PP and Andersen-Tawil syndrome are genetically heterogeneous. Over the past decade mutations in genes encoding three ion channels, CACN1AS, SCN4A and KCNJ2, have been identified and account for at least 70% of the identified cases of PP and several allelic disorders. No prospective clinical studies have followed sufficiently large cohorts with characterized molecular lesions to draw precise conclusions. We summarize current knowledge of the clinical diagnosis, molecular genetics, genotype-phenotype correlations, pathophysiology and treatment in the PPs. We focus on unresolved issues including (i) Are there additional ion channel defects in cases without defined mutations? (ii) What is the mechanism for depolarization-induced weakness in Hypo PP? and finally (iii) Will detailed electrophysiological studies be able to correctly identify specific channel mutations? Understanding the pathophysiology of the potassium-sensitive PPs ought to reduce genetic complexity, allow subjects to be stratified during future clinical trials and increase the likelihood of observing true clinical effects. Ideally, therapy for the PPs will prevent attacks, avoid permanent weakness and improve quality of life. Moreover, understanding the skeletal muscle channelopathies will hopefully lead to insights into the more common central nervous system channel diseases such as migraine and epilepsy.

Mexiletine block of disease-associated mutations in S6 segments of the human skeletal muscle Na(+) channel.

1. Over twenty different missense mutations in the alpha-subunit of the adult skeletal muscle Na(+) channel (hSkM1) have been identified as a cause of myotonia or periodic paralysis. We examined state-dependent mexiletine block for mutations involving the putative binding site in S6 segments (V445M, S804F, V1293I, V1589M and M1592V). Whole-cell Na(+) currents were measured from wild-type (WT) and mutant channels transiently expressed in HEK cells. 2. Use-dependent block (10 ms pulses to -10 mV, at 20 Hz) in 100 microM mexiletine was reduced modestly by mutations in IVS6 (V1589M, M1592V) and enhanced by the mutation in IS6 (V445M). For mutations in IIS6 (S804F) and IIIS6 (V1293I) use-dependent block was not statistically different from that of wild-type channels. 3. Resting-state block (10 ms pulses to -10 mV from -150 mV, at 0.1 Hz) of S6 mutants was comparable to that of WT (dissociation constant for resting channels, K(R) = 650 +/- 40 microM, n = 9). The S6 mutant with the greatest change in K(R) was V445M (K(R) = 794 +/- 45 microM, n = 5), but this difference was only marginally significant (P = 0.047). 4. A modified technique for estimating local anaesthetic affinity of inactivated channels was developed to reduce errors due to slow inactivation and to failure of drug binding to reach equilibrium. Mexiletine affinity for inactivated channels was reduced by mutations in IVS6 (V1589M: dissociation constant for the inactivated state (K(I)) = 44.7 microM; M1592V: K(I) = 40.0 microM) and increased by the mutation in IS6 (V445M: K(I) = 15.0 microM), compared to wild-type channels (K(I) = 28.3 microM). 5. We conclude that the disease-associated S6 mutations in domains I-IV cause at most a 2-fold change in inactivated state affinity and have even less of an effect on resting block. Model simulations show that the reduced use-dependent block of IVS6 mutants derives primarily from an increased off-rate at hyperpolarized potentials, whereas the enhanced use-dependent block of the IS6 mutant was due to a higher affinity for inactivated V445M channels.

A new mutation in a family with cold-aggravated myotonia disrupts Na(+) channel inactivation.

OBJECTIVE: To identify the molecular and physiologic abnormality in familial myotonia with cold sensitivity, hypertrophy, and no weakness. BACKGROUND: Sodium channel mutations were previously identified as the cause of several allelic disorders with varying combinations of myotonia and periodic paralysis. A three-generation family with dominant myotonia aggravated by cooling, but no weakness, was screened for mutations in the skeletal muscle sodium channel alpha-subunit gene (SCN4A). METHODS: Single-strand conformation polymorphism was used to screen all 24 exons of SCN4A and abnormal conformers were sequenced to confirm the presence of mutations. The functional consequence of a SCN4A mutation was explored by recording sodium currents from human embryonic kidney cells transiently transfected with an expression construct that was mutated to reproduce the genetic defect. RESULTS: A three-generation Italian family with myotonia is presented, in which a novel SCN4A mutation (leucine 266 substituted by valine, L266V) is identified. This change removes only a single methylene group from the 1,836-amino-acid protein, and is present in a region of the protein previously not known to be critical for channel function (domain I transmembrane segment 5). Electrophysiologic studies of the L266V mutation showed defects in fast inactivation, consistent with other disease-causing SCN4A mutations studied to date. Slow inactivation was not impaired. CONCLUSIONS: This novel mutation of the sodium channel indicates that a single carbon change in a transmembrane alpha-helix of domain I can alter channel inactivation and cause cold-sensitive myotonia.

MOD-1 is a serotonin-gated chloride channel that modulates locomotory behaviour in C. elegans.

The neurotransmitter and neuromodulator serotonin (5-HT) functions by binding either to metabotropic G-protein-coupled receptors (for example, 5-HT1, 5-HT2, 5-HT4 to 5-HT7), which mediate 'slow' modulatory responses through numerous second messenger pathways, or to the ionotropic 5-HT3 receptor, a non-selective cation channel that mediates 'fast' membrane depolarizations. Here we report that the gene mod-1 (for modulation of locomotion defective) from the nematode Caenorhabditis elegans encodes a new type of ionotropic 5-HT receptor, a 5-HT-gated chloride channel. The predicted MOD-1 protein is similar to members of the nicotinic acetylcholine receptor family of ligand-gated ion channels, in particular to GABA (gamma-aminobutyric acid)- and glycine-gated chloride channels. The MOD-1 channel has distinctive ion selectivity and pharmacological properties. The reversal potential of the MOD-1 channel is dependent on the concentration of chloride ions but not of cations. The MOD-1 channel is not blocked by calcium ions or 5-HT3a-specific antagonists but is inhibited by the metabotropic 5-HT receptor antagonists mianserin and methiothepin. mod-1 mutant animals are defective in a 5-HT-mediated experience-dependent behaviour and are resistant to exogenous 5-HT, confirming that MOD-1 functions as a 5-HT receptor in vivo.

COOH-terminal truncated alpha(1S) subunits conduct current better than full-length dihydropyridine receptors.

Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca(2+) channels and as voltage sensors for coupling membrane depolarization to release of Ca(2+) from the sarcoplasmic reticulum. In skeletal muscle, the principal or alpha(1S) subunit occurs in full-length ( approximately 10% of total) and post-transcriptionally truncated ( approximately 90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized alpha(1S) subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (alpha(1S)) or truncated (alpha(1SDeltaC)) alpha subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Q(max) 80-150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in alpha(1S), unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba(2+) currents were small relative to Q(max). The truncated alpha(1SDeltaC) protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the alpha(1S) subunit conduct the majority of the L-type Ca(2+) current in skeletal muscle. Our data also suggest that the carboxyl terminus of the alpha(1S) subunit modulates the coupling between charge movement and channel opening.

Rapid and slow voltage-dependent conformational changes in segment IVS6 of voltage-gated Na(+) channels.

Mutations in segment IVS6 of voltage-gated Na(+) channels affect fast-inactivation, slow-inactivation, local anesthetic action, and batrachotoxin (BTX) action. To detect conformational changes associated with these processes, we substituted a cysteine for a valine at position 1583 in the rat adult skeletal muscle sodium channel alpha-subunit, and examined the accessibility of the substituted cysteine to modification by 2-aminoethyl methanethiosulfonate (MTS-EA) in excised macropatches. MTS-EA causes an irreversible reduction in the peak current when applied both internally and externally, with a reaction rate that is strongly voltage-dependent. The rate increased when exposures to MTS-EA occurred during brief conditioning pulses to progressively more depolarized voltages, but decreased when exposures occurred at the end of prolonged depolarizations, revealing two conformational changes near site 1583, one coupled to fast inactivation, and one tightly associated with slow inactivation. Tetraethylammonium, a pore blocker, did not affect the reaction rate from either direction, while BTX, a lipophilic activator of sodium channels, completely prevented the modification reaction from occurring from either direction. We conclude that there are two inactivation-associated conformational changes in the vicinity of site 1583, that the reactive site most likely faces away from the pore, and that site 1583 comprises part of the BTX receptor.

Spectrum of sodium channel disturbances in the nondystrophic myotonias and periodic paralyses.

Several heritable forms of myotonia and periodic paralysis are caused by missense mutations in the voltage-gated sodium channel of skeletal muscle. Mutations produce gain-of-function defects, either disrupted inactivation or enhanced activation. Both defects result in too much inward Na current which may either initiate pathologic bursts of action potentials (myotonia) or cause flaccid paralysis by depolarizing fibers to a refractory inexcitable state. Myotonic stiffness and periodic paralysis occur as paroxysmal attacks often triggered by environmental factors such as serum K+, cold, or exercise. Many gaps remain in our understanding of the interactions between genetic predisposition and these environmental influences. Targeted gene manipulation in animals may provide the tools to fill in these gaps.

The human skeletal muscle Na channel mutation R669H associated with hypokalemic periodic paralysis enhances slow inactivation.

Missense mutations of the human skeletal muscle voltage-gated Na channel (hSkM1) underlie a variety of diseases, including hyperkalemic periodic paralysis (HyperPP), paramyotonia congenita, and potassium-aggravated myotonia. Another disorder of sarcolemmal excitability, hypokalemic periodic paralysis (HypoPP), which is usually caused by missense mutations of the S4 voltage sensors of the L-type Ca channel, was associated recently in one family with a mutation in the outermost arginine of the IIS4 voltage sensor (R669H) of hSkM1 (Bulman et al., 1999). Intriguingly, an arginine-to-histidine mutation at the homologous position in the L-type Ca(2+) channel (R528H) is a common cause of HypoPP. We have studied the gating properties of the hSkM1-R669H mutant Na channel experimentally in human embryonic kidney cells and found that it has no significant effects on activation or fast inactivation but does cause an enhancement of slow inactivation. R669H channels exhibit an approximately 10 mV hyperpolarized shift in the voltage dependence of slow inactivation and a twofold to fivefold prolongation of recovery after prolonged depolarization. In contrast, slow inactivation is often disrupted in HyperPP-associated Na channel mutants. These results demonstrate that, in R669H-associated HypoPP, enhanced slow inactivation does not preclude, and may contribute to, prolonged attacks of weakness and add support to previous evidence implicating the IIS4 voltage sensor in slow-inactivation gating.

Effects of mutations causing hypokalaemic periodic paralysis on the skeletal muscle L-type Ca2+ channel expressed in Xenopus laevis oocytes.

1. A truncated form of the rabbit alpha1S Ca2+ channel subunit (alpha1SDeltaC) was expressed with the beta1b, alpha2delta and gamma auxiliary subunits in Xenopus laevis oocytes. After 5-7 days, skeletal muscle L-type currents were measured (469 +/- 48 nA in 10 mM Ba2+). All three of the auxiliary subunits were necessary to record significant L-type current. A rapidly inactivating, dihydropyridine-insensitive endogenous Ba2+ current was observed in oocytes expressing the auxiliary subunits without an exogenous alpha subunit. Expression of full-length alpha1S gave 10-fold smaller currents than the truncated form. 2. Three missense mutations causing hypokalaemic periodic paralysis (R528H in domain II S4 of the alpha1S subunit; R1239H and R1239G in domain IV S4) were introduced into alpha1SDeltaC and expressed in oocytes. L-type current was separated from the endogenous current by nimodipine subtraction. All three of the mutations reduced L-type current amplitude ( approximately 40 % for R528H, approximately 60-70 % for R1239H and R1239G). 3. The disease mutations altered the activation properties of L-type current. R528H shifted the G(V) curve approximately 5 mV to the left and modestly reduced the voltage dependence of the activation time constant, tauact. R1239H and R1239G shifted the G(V) curve approximately 5-10 mV to the right and dramatically slowed tauact at depolarized test potentials. 4. The voltage dependence of steady-state inactivation was not significantly altered by any of the disease mutations. 5. Wild-type and mutant L-type currents were also measured in the presence of (-)-Bay K8644, which boosted the amplitude approximately 5- to 7-fold. The effects of the mutations on the position of the G(V) curve and the voltage dependence of tauact were essentially the same as in the absence of agonist. Bay K-enhanced tail currents were slowed by R528H and accelerated by R1239H and R1239G. 6. We conclude that the domain IV mutations R1239H and R1239G have similar effects on the gating properties of the skeletal muscle L-type Ca2+ channel expressed in Xenopus oocytes, while the domain II mutation R528H has distinct effects. This result implies that the location of the substitutions is more important than their degree of conservation in determining their biophysical consequences.

Defective slow inactivation of sodium channels contributes to familial periodic paralysis.

OBJECTIVE: To evaluate the effects of missense mutations within the skeletal muscle sodium (Na) channel on slow inactivation (SI) in periodic paralysis and related myotonic disorders. BACKGROUND: Na channel mutations in hyperkalemic periodic paralysis and the nondystrophic myotonias interfere with the normally rapid inactivation of muscle Na currents following an action potential. This defect causes persistent inward Na currents that produce muscle depolarization, myotonia, or onset of weakness. Distinct from fast inactivation is the process called SI, which limits availability of Na channels on a time scale of seconds to minutes, thereby influencing muscle excitability. METHODS: Human Na channel cDNAs containing mutations associated with paralytic and nonparalytic phenotypes were transiently expressed in human embryonic kidney cells for whole-cell Na current recording. Extent of SI over a range of conditioning voltages (-120 to +20 mV) was defined as the fraction of Na current that failed to recover within 20 ms at - 100 mV. The time course of entry to SI at -30 mV was measured using a conditioning pulse duration of 20 ms to 60 seconds. Recovery from SI at -100 mV was assessed over 20 ms to 10 seconds. RESULTS: The two most common hyperkalemic periodic paralysis (HyperPP) mutations responsible for episodic attacks of weakness or paralysis, T704M and M1592V, showed clearly impaired SI, as we and others have observed previously for the rat homologs of these mutations. In addition, a new paralysis-associated mutant, I693T, with cold-induced weakness, exhibited a comparable defect in SI. However, SI remained intact for both the HyperPP/paramyotonia congenita (PMC) mutant, A1156T, and the nonparalytic potassium-aggravated myotonia (PAM) mutant, V1589M. CONCLUSIONS: SI is defective in a subset of mutant Na channels associated with episodic weakness (HyperPP or PMC) but remains intact for mutants studied so far that cause myotonia without weakness (PAM).

Enhanced slow inactivation by V445M: a sodium channel mutation associated with myotonia.

Over 20 different missense mutations in the alpha subunit of the adult skeletal muscle Na channel have been identified in families with either myotonia (muscle stiffness) or periodic paralysis, or both. The V445M mutation was recently found in a family with myotonia but no weakness. This mutation in transmembrane segment IS6 is novel because no other disease-associated mutations are in domain I. Na currents were recorded from V445M and wild-type channels transiently expressed in human embryonic kidney cells. In common with other myotonic mutants studied to date, fast gating behavior was altered by V445M in a manner predicted to increase excitability: an impairment of fast inactivation increased the persistent Na current at 10 ms and activation had a hyperpolarized shift (4 mV). In contrast, slow inactivation was enhanced by V445M due to both a slower recovery (10 mV left shift in beta(V)) and an accelerated entry rate (1.6-fold). Our results provide additional evidence that IS6 is crucial for slow inactivation and show that enhanced slow inactivation cannot prevent myotonia, whereas previous studies have shown that disrupted slow inactivation predisposes to episodic paralysis.

The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels.

Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel alpha subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.

Gating of the L-type Ca channel in human skeletal myotubes: an activation defect caused by the hypokalemic periodic paralysis mutation R528H.

The skeletal muscle L-type Ca channel serves a dual role as a calcium-conducting pore and as the voltage sensor coupling t-tubule depolarization to calcium release from the sarcoplasmic reticulum. Mutations in this channel cause hypokalemic periodic paralysis (HypoPP), a human autosomal dominant disorder characterized by episodic failure of muscle excitability that occurs in association with a decrease in serum potassium. The voltage-dependent gating of L-type Ca channels was characterized by recording whole-cell Ca currents in myotubes cultured from three normal individuals and from a patient carrying the HypoPP mutation R528H. We found two effects of the R528H mutation on the L-type Ca current in HypoPP myotubes: (1) a mild reduction in current density and (2) a significant slowing of the rate of activation. We also measured the voltage dependence of steady-state L-type Ca current inactivation and characterized, for the first time in a mammalian preparation, the kinetics of both entry into and recovery from inactivation over a wide range of voltages. The R528H mutation had no effect on the kinetics or voltage dependence of inactivation.

Human sodium channel gating defects caused by missense mutations in S6 segments associated with myotonia: S804F and V1293I.

1. Missense mutations in the alpha-subunit of the human skeletal muscle sodium channel (hSkM1) have been detected in some heritable forms of myotonia. By recording Na+ currents from cells transfected with cDNA encoding either wild-type or mutant hSkM1, we characterized the functional consequences of two myotonia-associated mutations that lie at the cytoplasmic end of the sixth transmembrane segment in domain II (S804F) or domain III (V1293I). 2. Both mutations caused modest, but unequivocal, alterations in the voltage-dependent gating behaviour of hSkM1. For S804F, the abnormalities were limited to fast inactivation: the persistent Na+ current at the end of a 50 ms depolarization was increased 3-fold, the rate of inactivation from the open state was slowed 2-fold, and the voltage dependence of fast inactivation (h) was shifted by +3 mV. V1293I also disrupted fast inactivation, as evidenced by a 3-fold faster rate of recovery at hyperpolarized potentials (-70 mV). Activation was altered as well for V1293I: the voltage dependence was shifted by -6 mV (hyperpolarized). 3. Slow inactivation was not altered by S804F or V1293I. 4. We conclude that S804F and V1293I are not benign polymorphisms. Either mutation causes detectable alterations in channel gating and, in model simulations, the magnitude of the defects is sufficient to produce runs of myotonic discharges.

Slow inactivation does not affect movement of the fast inactivation gate in voltage-gated Na+ channels.

Voltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (microl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (hinfinity) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

A proposed mutation, Val781Ile, associated with hyperkalemic periodic paralysis and cardiac dysrhythmia is a benign polymorphism.

Twenty different point mutations have been identified in the gene coding for the alpha subunit of the adult skeletal muscle sodium channel in families with hyperkalemic periodic paralysis, paramyotonia congenita, and the potassium-aggravated myotonias. One novel mutation (Val(781)Ile) was reported in an adopted boy with potassium-sensitive weakness and cardiac dysrhythmia. The confidence in establishing this rare amino acid substitution as a causative mutation was limited by the absence of family members for segregation analysis. Functional expression studies herein show that Val(781)Ile is most likely a benign polymorphism and not a disease-associated mutation.

From mutation to myotonia in sodium channel disorders.

Hyperkalemic periodic paralysis, paramyotonia congenita, and the potassium-aggravated myotonias are all caused by point mutations in the alpha-subunit of a sodium channel expressed selectively in skeletal muscle. This review updates the growing list of genotype-phenotype correlations for these mutations and summarizes the alterations in channel function they produce. A toxin-based in vitro model demonstrates that subtle defects in sodium channel inactivation are sufficient to cause myotonia and computer modeling suggests that specific types of inactivation defect may predispose to paralysis or myotonia.

Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis.

Several heritable forms of myotonia and hyperkalemic periodic paralysis (HyperPP) are caused by missense mutations in the alpha subunit of the skeletal muscle Na channel (SkM1). These mutations impair fast inactivation or shift activation toward hyperpolarized potentials, inducing persistent Na currents that may cause muscle depolarization, myotonia, and onset of weakness. It has been proposed that the aberrant Na current and resulting weakness will be sustained only if Na channel slow inactivation is also impaired. We therefore measured slow inactivation for wild-type and five mutant Na channels constructed in the rat skeletal muscle isoform (rSkM1) and expressed in HEK cells. Two common HyperPP mutations (T698M in domain II-S5 and M1585V in IV-S6) had defective slow inactivation. This defect reduced use-dependent inhibition of Na currents elicited during 50-Hz stimulation. A rare HyperPP mutation (M1353V in IV-S1) and mutations within the domain III-IV linker that cause myotonia (G1299E) or myotonia plus weakness (T1306M) did not impair slow inactivation. We also observed that slow inactivation of wild-type rSkM1 was incomplete; therefore it is possible that stable membrane depolarization and subsequent muscle weakness may be caused solely by defects in fast inactivation or activation. Model simulations showed that abnormal slow inactivation, although not required for expression of a paralytic phenotype, may accentuate muscle membrane depolarization, paralysis, and sensitivity to hyperkalemia.