Structural basis for gating pore current in periodic paralysis.

Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness1,2. They are caused by single mutations in positively charged residues ('gating charges') in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Nav1.4 or the calcium channel Cav1.11,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis 5 owing to cation leak in both activated and inactivated states 6 . Here we present high-resolution structures of the model bacterial sodium channel NavAb with the analogous gating-charge mutations7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of NavAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.

Andersen cardiodysrhythmic periodic paralysis with KCNJ2 mutations: a novel mutation in the pore selectivity filter residue.

Andersen cardiodysrhythmic periodic paralysis or Andersen-Tawil syndrome includes the distinct clinical features of periodic paralysis, cardiac arrhythmia, and facial and skeletal dysmorphisms and exhibits autosomal dominant inheritance. Mutations in the KCNJ2 gene, which encodes the human inward rectifier potassium channel Kir2.1, have been identified in the majority of cases. Despite well-established clinical and molecular characteristics, treatment is still case oriented, and timely diagnosis could be delayed because of the low incidence and phenotypic heterogeneity of this disease. This article describes the clinical and molecular features of 3 cases of Andersen-Tawil syndrome in 2 families. One of the mutations (G144D) was located in the pore selectivity filter residue (which is mutated recurrently) and was considered novel. Intermittent muscle weakness in childhood warrants careful evaluation of cardiac dysrhythmia and skeletal anomalies.

[The construction and preliminary investigation of the cell model of a novel mutation R675Q in the SCN4A gene identified in a Chinese family with normokalemic periodic paralysis].

OBJECTIVE: To construct and investigate the cell model of a novel mutation R675Q in the skeletal muscle Na channel type 4 alpha subunit gene (SCN4A) identified from a Chinese family with normokalemic periodic paralysis. METHODS: cDNA encoding the adult isoform of SCN4A was used as a template for in vitro site-directed mutagenesis by PCR method. The mutated plasmid was transiently transfected into HEK-293 cells by calcium phosphate precipitation. Twenty four and 48 hours after transfection, the expression level of SCN4A was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Whole cell voltage-clamp recording was used to study the current of sodium channels. RESULTS: The site-mutagenesis of the plasmid was confirmed by sequencing. The expression of SCN4A gene was significantly elevated 24 h and 48 h after transfection. The relative current of R675Q is smaller than that of wide type before reaching peak current under the same test voltage, but larger than that of wild type current after reaching peak current. They both had the largest peak current under 0 mV test pulse. CONCLUSION: A cell model of normokalemic periodic paralysis was successfully constructed. The R675Q mutation of the SCN4A gene enhances the activation and inactivation of the sodium channel, and the S4 transmembrane segment may have intimate relationship with the attack of weakness in normoKPP patients.

The dominant cold-sensitive Out-cold mutants of Drosophila melanogaster have novel missense mutations in the voltage-gated sodium channel gene paralytic.

Here we report the molecular characterization of Out-cold (Ocd) mutants of Drosophila melanogaster, which produce a dominant, X-linked, cold-sensitive paralytic phenotype. From its initial 1.5-Mb cytological location within 13F1-16A2, P-element and SNP mapping reduced the Ocd critical region to <100 kb and to six candidate genes: hangover, CG9947, CG4420, eIF2a, Rbp2, and paralytic (para). Complementation testing with para null mutations strongly suggests Ocd and para are allelic, as does gene rescue of Ocd semilethality with a wild-type para transgene. Pesticide resistance and electrophysiological phenotypes of Ocd mutants support this conclusion. The para gene encodes a voltage-gated sodium channel. Sequencing the Ocd lines revealed mutations within highly conserved regions of the para coding sequence, in the transmembrane segment S6 of domain III (I1545M and T1551I), and in the linker between domains III and IV (G1571R), the location of the channel inactivation gate. The G1571R mutation is of particular interest as mutations of the orthologous residue (G1306) in the human skeletal muscle sodium channel gene SCN4A are associated with cases of periodic paralysis and myotonia, including the human cold-sensitive disorder paramyotonia congenita. The mechanisms by which sodium channel mutations cause cold sensitivity are not well understood. Therefore, in the absence of suitable vertebrate models, Ocd provides a system in which genetic, molecular, physiological, and behavioral tools can be exploited to determine mechanisms underlying sodium channel periodic paralyses.

Muscle membrane excitability after exercise in thyrotoxic periodic paralysis and thyrotoxicosis without periodic paralysis.

We evaluated whether the paralytic attacks in thyrotoxic periodic paralysis (TPP) are primarily due to the abnormal excitability of the muscle membrane caused by a preexisting latent abnormality or to the effects of thyroid hormone. The prolonged exercise (PE) test was used to evaluate muscle membrane excitability in 21 patients with TPP and 11 patients with thyrotoxicosis without paralytic attacks (Tw/oPP) in the hyperthyroid state. The PE tests were compared between the hyperthyroid and euthyroid states in five of the TPP and three of the Tw/oPP patients. Compared to 20 healthy subjects, a significant increase in compound muscle action potential (CMAP) amplitudes immediately after exercise and a significant time-dependent gradual decline in CMAP amplitudes starting from 20 min after exercise were observed in the TPP patients. A significant decline in CMAP amplitudes was also observed in the Tw/oPP patients but only at 50 min after exercise. All of the TPP and Tw/oPP patients had a tendency to improve in the euthyroid state; the PE tests remained abnormal only in the TPP patients. Paralytic attacks in TPP patients are due primarily to a preexisting latent abnormal excitability of the muscle membrane, possibly genetic in origin.

Phosphorylation and protonation of neighboring MiRP2 sites: function and pathophysiology of MiRP2-Kv3.4 potassium channels in periodic paralysis.

MinK-related peptide 2 (MiRP2) and Kv3.4 subunits assemble in skeletal muscle to create subthreshold, voltage-gated potassium channels. MiRP2 acts on Kv3.4 to shift the voltage dependence of activation, speed recovery from inactivation, suppress cumulative inactivation and increase unitary conductance. We previously found an R83H missense mutation in MiRP2 that segregated with periodic paralysis in two families and diminished the effects of MiRP2 on Kv3.4. Here we show that MiRP2 has a single, functional PKC phosphorylation site at serine 82 and that normal MiRP2-Kv3.4 function requires phosphorylation of the site. The R83H variant does not prevent PKC phosphorylation of neighboring S82; rather, the change shifts the voltage dependence of activation and endows MiRP2-Kv3.4 channels with sensitivity to changes in intracellular pH across the physiological range. Thus, current passed by single R83H channels decreases as internal pH is lowered (pK(a) approximately 7.3, consistent with histidine protonation) whereas wild-type channels are largely insensitive. These findings identify a key regulatory domain in MiRP2 and suggest a mechanistic link between acidosis and episodes of periodic paralysis.

[Neuromuscular hereditary channelopathies: non-dystrophic myotonias, paramyotonias and periodic paralysis]. / Canalopatias hereditarias neuromusculares: miotonías no distróficas, paramiotonías y parálisis periódicas.

INTRODUCTION: The ionic channels are complex glycoprotein structures, which cross the lipidic cellular membrane and allow the passage of electrically charged ions from one side of it to the other, thanks to the electrochemical gradient. A channelopathy is a disorder due to anomalous function of the ionic channels. DEVELOPMENT: In this study we analyze particularly the hereditary channelopathies with neuromuscular involvement non dystrophic myotonia, paramyotonias and periodic paralysis, and classify the clinical, physiopathological, molecular, genetic and therapeutic aspects. As far as possible we have divided the different conditions according to the channel involved, due to mutations which affect the sodium, calcium, chloride and potassium channels. We have also included neuromyotonic phenomena which are probably caused by channelopathies. CONCLUSIONS: Probably it will not be long before many of the conditions considered in this article have a better physiopathological explanation, more specific diagnostic procedures and a more rational approach to treatment.

A double mutation in families with periodic paralysis defines new aspects of sodium channel slow inactivation.

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant skeletal muscle disorder caused by single mutations in the SCN4A gene, encoding the human skeletal muscle voltage-gated Na(+) channel. We have now identified one allele with two novel mutations occurring simultaneously in the SCN4A gene. These mutations are found in two distinct families that had symptoms of periodic paralysis and malignant hyperthermia susceptibility. The two nucleotide transitions predict phenylalanine 1490-->leucine and methionine 1493-->isoleucine changes located in the transmembrane segment S5 in the fourth repeat of the alpha-subunit Na(+) channel. Surprisingly, this mutation did not affect fast inactivation parameters. The only defect produced by the double mutant (F1490L-M1493I, expressed in human embryonic kidney 293 cells) is an enhancement of slow inactivation, a unique behavior not seen in the 24 other disease-causing mutations. The behavior observed in these mutant channels demonstrates that manifestation of HyperKPP does not necessarily require disruption of slow inactivation. Our findings may also shed light on the molecular determinants and mechanism of Na(+) channel slow inactivation and help clarify the relationship between Na(+) channel defects and the long-term paralytic attacks experienced by patients with HyperKPP.

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.

Activation and inactivation of the voltage-gated sodium channel: role of segment S5 revealed by a novel hyperkalaemic periodic paralysis mutation.

Hyperkalaemic periodic paralysis, paramyotonia congenita, and potassium-aggravated myotonia are three autosomal dominant skeletal muscle disorders linked to the SCN4A gene encoding the alpha-subunit of the human voltage-sensitive sodium channel. To date, approximately 20 point mutations causing these disorders have been described. We have identified a new point mutation, in the SCN4A gene, in a family with a hyperkalaemic periodic paralysis phenotype. This mutation predicts an isoleucine-to-phenylalanine substitution at position 1495 located in the transmembrane segment S5 in the fourth homologous domain of the human alpha-subunit sodium channel. Introduction of the I1495F mutation into the wild-type channels disrupted the macroscopic current inactivation decay and shifted both steady-state activation and inactivation to the hyperpolarizing direction. The recovery from fast inactivation was slowed, and there was no effect on channel deactivation. Additionally, a significant enhancement of slow inactivation was observed in the I1495F mutation. In contrast, the T704M mutation, a hyperkalaemic periodic paralysis mutation located in the cytoplasmic interface of the S5 segment of the second domain, also shifted activation in the hyperpolarizing direction but had little effect on fast inactivation and dramatically impaired slow inactivation. These results, showing that the I1495F and T704M hyperkalaemic periodic paralysis mutations both have profound effects on channel activation and fast-slow inactivation, suggest that the S5 segment maybe in a location where fast and slow inactivation converge.

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).

Impairment of skeletal muscle adenosine triphosphate-sensitive K+ channels in patients with hypokalemic periodic paralysis.

The adenosine triphosphate (ATP)-sensitive K+ (KATP) channel is the most abundant K+ channel active in the skeletal muscle fibers of humans and animals. In the present work, we demonstrate the involvement of the muscular KATP channel in a skeletal muscle disorder known as hypokalemic periodic paralysis (HOPP), which is caused by mutations of the dihydropyridine receptor of the Ca2+ channel. Muscle biopsies excised from three patients with HOPP carrying the R528H mutation of the dihydropyridine receptor showed a reduced sarcolemma KATP current that was not stimulated by magnesium adenosine diphosphate (MgADP; 50-100 microM) and was partially restored by cromakalim. In contrast, large KATP currents stimulated by MgADP were recorded in the healthy subjects. At channel level, an abnormal KATP channel showing several subconductance states was detected in the patients with HOPP. None of these were surveyed in the healthy subjects. Transitions of the KATP channel between subconductance states were also observed after in vitro incubation of the rat muscle with low-K+ solution. The lack of the sarcolemma KATP current observed in these patients explains the symptoms of the disease, i.e., hypokalemia, depolarization of the fibers, and possibly the paralysis following insulin administration.

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.

Impaired slow inactivation in mutant sodium channels.

Hyperkalemic periodic paralysis (HyperPP) is a disorder in which current through Na+ channels causes a prolonged depolarization of skeletal muscle fibers, resulting in membrane inexcitability and muscle paralysis. Although HyperPP mutations can enhance persistent sodium currents, unaltered slow inactivation would effectively eliminate any sustained currents through the mutant channels. We now report that rat skeletal muscle channels containing the mutation T698M, which corresponds to the human T704M HyperPP mutation, recover very quickly from prolonged depolarizations. Even after holding at -20 mV for 20 min, approximately 25% of the maximal sodium current is available subsequent to a 10-ms hyperpolarization (-100 mV). Under the same conditions, recovery is less than 3% in wild-type channels and in the F1304Q mutant, which has impaired fast inactivation. This effect of the T698M mutation on slow inactivation, in combination with its effects on activation, is expected to result in persistent currents such as that seen in HyperPP muscle.

The skeletal muscle sodium and chloride channel diseases.

The cause of several familial muscular diseases have recently been linked to mutations within skeletal muscle sodium and chloride channel genes. Thomsen's and Becker's diseases are autosomal dominant and recessive, respectively, and are caused by at least seven different mutations in the CLCN1 (ClC-1) skeletal muscle chloride channel gene on chromosome 7q35. Hyperkalaemic periodic paralysis, paramyotonia congenita and a small heterogeneous group of related 'pure' myotonias are autosomal dominant disorders and are due to at least 16 different mutations in the SCN4A (SkM1) adult skeletal muscle sodium channel gene on chromosome 17q23-25. There is generally little correlation between the position of a mutation in the channel and the phenotype. Indeed, identical sodium channel mutations in unrelated subjects and sometimes in different members of the same family can have different clinical expressions. It seems, however, that mutations of the inactivation gate (ID3-4 loop) of the sodium channel tend to produce paramyotonia or pure, sometimes severe, myotonia and respond most favourably to the same medications (tocainide and mexiletine). The structure and polarity of substituted amino acids at a mutation site, especially in highly evolutionally conserved regions of the gene, are undoubtedly important to the expression of a channel disease and may partly explain phenotypic variability. In addition, genetic polymorphisms elsewhere, either in the gene or other channel-related loci, and the net effect of other types of muscle ion channels on the electrical potential of the plasma membrane probably contribute to disease expression.