Muscle Sodium Research Articles

The relationship of cerebral oximeter and postoperative cognition function of geriatric patients under intravenous anesthesia using creatine phosphate disodium salt

Objective To discuss the relationship of perioperative regional cerebral saturation of oxygenation (rSO2) and postoperative cognition function alteration of geriatric patients under intravenous anesthesia using creatine phosphate disodium salt,and to establish the clinical practical guides.Methods Sixty ASA Ⅰ-Ⅱ patients,aged＞60 y scheduled for selective abdominal surgeries or surgeries on lower limb were enrolled in this study.We used a completely randomized grouping design for this study and the 20 patients were divided into 3 groups:creatine phosphate disodium salt group 1 (C1),creatine phosphate disodium salt group 2 (C2),Control group (C).All patients were not premeditated with atropine 0.5 mg until entering the operation room.Anesthesia was induced with intravenous infusion of propofol,fentanyl and cisatracurium slowly,The anesthesia was maintained by intravenous propofol infusion in target controlled infusion target controlled infusion (TCI) (3 μg) and remifentanyl (0.2 μg·kg-1·min-1-0.4 μg·kg-1·min-1) infusion,intravenous bolus cisatracurium (0.07 mg/kg-0.10 mg/kg).After tracheal intubation,all patients were mechanically ventilated with expiration carbon dioxide pressure (PERCO2) at normal scale.The rSO2 was continuously monitored and recorded in operations.The mini-mental state examination (MMSE),trail-making test and grooved pegboard test were adopted to access cognitive function 24 h before surgery and 4,8,12,24 h after surgery.Results ① There were no significantly difference of general status among the three groups(P＞0.05).② The scales of MMSE,trail-making test and grooved pegboard test were not different 24 h before operations among three groups (P＞0.05).Compared with scales of MMSE before operation,4,8,12 h after operation in all patients were significantly lower (P＜0.05).Three groups of patients trail-making test and the grooved pegboard test time comparison experiment found:there were no difference between preoperative and postoperative testing time (P＞0.05),which time of patients in group C2 completed the task significantly shorter than group C1,the control group of patients needed more time to complete the task.Comparison of postoperative cognitive function changes after 4 h,cognitive function changes occurred in the control group of patients with 3 patients (15％),creatine phosphate (C2) of patients in group 1 patient (5％),creatine phosphate sodium (C1) patients,2 patients (10％).After 8 h,patients in the control group 2 patients (10％),creatine phosphate (C1) patients in group 1 patients (5％),phosphoric acid muscle sodium (C2) cognitive function in patients recovered.Postoperative 12 h,patients in the control group only one case (5％),creatine phosphate groups (group C1 and C2) cognitive function in patients recovered,after 24 h,cognitive function of all patients recovered,the delirium did not occur in all patients entered the study.(③ The scales of cognitive tests were higher in group C2 and group C1 than C (P＜0.01).The numbers of rSO2 in the three groups were identical (P＞0.05).Conclusions Creatine phosphate disodium salt can reduce the occurrence of postoperative cognitive dysfunction in geriatric patients combined the intravenous and inhalational anesthesia.The results may relate to its neural protection effects,unique natural energy supply and increasing central neural system tolerance of ischemia and hypoxia. Key words: Geriatric patients; Postoperative cognition function; Regional cerebral saturation of oxygenation; Creatine phosphate disodium salt

Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.

Normokalemic periodic paralysis with involuntary movements and generalized epilepsy associated with two novel mutations in SCN4A gene

Mutations of skeletal muscle sodium channel a subunit (SCN4A) gene are associated with a group of allelic diseases, including periodic paralysis, paramyotonia congenital, sodium channel myotonia, and congenital myasthenic syndrome. Periodic paralysis is characterized by episodic attacks of flaccid weakness with the fluctuation of serum potassium, which are usually limited to skeletal muscles owing to tissue specific expression of channel isoforms. However, periodic paralysis is occasionally concomitant with cardiac arrhythmia, essential tremor, and epilepsy. Here we described a case of normokalemic periodic paralysis with involuntary movements and generalized epilepsy associated with two novel heterozygous missense mutations in SCN4A gene.

Parallel evolution of tetrodotoxin resistance in three voltage-gated sodium channel genes in the garter snake Thamnophis sirtalis.

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav’s, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1–1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood–brain barrier. We also report the exon–intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations.

Hyperkalemic Periodic Paralysis Caused by a Mutation in the Sodium Channel SCN4A Gene

Hyperkalemic periodic paralysis (HyperPP) is an autosomal dominant muscle sodium channelopathy characterized by recurrentepisode of reversible paralysis with concomitant hyperkalemia. The diagnosis of HyperPP is suggested by a history of attacks ofparalysis, positive family history, and the presence of myotonia. A 19-year-old man presented with recurrent generalized limbweakness since childhood. The paralysis often followed by fatigue, exercise, and fasting. His parents were clinically unaffectedand had never experienced paralytic symptoms. Electromyographic evaluation demonstrated myotonic discharge. Directsequencing of SCN4A exon 24 revealed a heterozygous A>G transition at nucleotide 4774, resulting in the substitution of amethionine by a valine at codon 1592 (Met1592Val). We report a patient with HyperPP confirmed by Met1592Val mutation inSCN4A gene.

Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains.

Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation-pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in NaV VSDs.

Roles of Hyp Residues in the Folding and Activity of μ-Conotoxin GIIIA, a Peptide Blocker of Muscle Sodium Channels

Attempts were made to synthesize seven analogs of µ-conotoxin GIIIA, a specific blocker of muscle sodium channels, by replacing the three Hyp residues (Hyp6, Hyp7, and Hyp17) with various amino acids. Replacement with Ala residue at these positions resulted in a very low isolation yield, suggesting that these three Hyp residues are essential for the folding of the molecule. CD spectra of the synthesized analogs suggest that, once synthesized, the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the hydroxyl group at side chains of Hyp residues are not essential for the activity.

N-Aryl-2,6-dimethylbenzamides, a New Generation of Tocainide Analogues as Blockers of Skeletal Muscle Voltage-Gated Sodium Channels

On the basis of a 3D-QSAR study, a new generation of tocainide analogues were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. Data obtained by screening new compounds by means of Hille-Campbell Vaseline gap voltage-clamp recordings showed that the elongation of the alkyl chain and the introduction of lipophilic and sterically hindered groups on the amino function enhance both potency and use-dependent block. The results provide additional indications about the structural requirement of pharmacophores for further increasing potency and state-dependent block and allowed us to identify a new tocainide analogue (6f) with a favorable pharmacodynamic profile to be proposed as a valid candidate for studies aimed at evaluating its usefulness in the treatment of myotonias.

Preclinical evaluation of marketed sodium channel blockers in a rat model of myotonia discloses promising antimyotonic drugs

Although the sodium channel blocker mexiletine is considered the first-line drug in myotonia, some patients experiment adverse effects, while others do not gain any benefit. Other antimyotonic drugs are thus needed to offer mexiletine alternatives. In the present study, we used a previously-validated rat model of myotonia congenita to compare six marketed sodium channel blockers to mexiletine. Myotonia was induced in the rat by injection of anthracen-9-carboxylic acid, a muscle chloride channel blocker. The drugs were given orally and myotonia was evaluated by measuring the time of righting reflex. The drugs were also tested on sodium currents recorded in a cell line transfected with the human skeletal muscle sodium channel hNav1.4 using patch-clamp technique. In vivo, carbamazepine and propafenone showed antimyotonic activity at doses similar to mexiletine (ED50 close to 5mg/kg); flecainide and orphenadrine showed greater potency (ED50 near 1mg/kg); lubeluzole and riluzole were the more potent (ED50 near 0.1mg/kg). The antimyotonic activity of drugs in vivo was linearly correlated with their potency in blocking hNav1.4 channels in vitro. Deviation was observed for propafenone and carbamazepine, likely due to pharmacokinetics and multiple targets. The comparison of the antimyotonic dose calculated in rats with the current clinical dose in humans strongly suggests that all the tested drugs may be used safely for the treatment of human myotonia. Considering the limits of mexiletine tolerability and the occurrence of non-responders, this study proposes an arsenal of alternative drugs, which may prove useful to increase the quality of life of individuals suffering from non-dystrophic myotonia. Further clinical trials are warranted to confirm these results.

Congruent Pattern of Accessibility within the Pore of a Voltage-Gated Na+ Channel

Voltage-gated ion channels open a gate in response to membrane depolarization which allows ions to pass through the transmembrane pore of the channel. MTSET accessibility studies within the Shaker potassium channel revealed that K+ permeation is controlled by an intracellular gate, whereas Cd2+ and Ag+ accessibility measurements in the cyclic nucleotide-gated channel suggests that this process occurs at the selectivity filter. Modification data of cysteine residues introduced into the DIV S6 of a fast-inactivation removed voltage-gated sodium channel (VGSC) by MTSET indicate that sodium channel gating is also regulated by an intracellular gate. However, unlike the Shaker potassium channel, VGSCs are not composed of four identical subunits. Despite sequence similarity between each S6 helix, the multi-domain nature of VGSCs implies that there may be asymmetry within the pore that may have consequences in regard to channel gating. Thus, we sought to determine if analogous positions within the S6 helices of a VGSC act together to form an intracellular gate which occludes the pore while channels are closed. We scanned the MTSET accessibility of substituted cysteines in the pore lining helices of the first three domains (DI-DIII) in the rat skeletal muscle sodium channel. The modification data at these sites, selected on the basis of sequence alignment and MTSET accessibility data of DIV-S6, confirms that DII and DIII follow the same pattern of accessibility as that of DIV; however, the extent of block is quite varied across each domain. Along with DI accessibility data, these findings will be discussed in the context of pore gating in related voltage-gated ion channels.

A Novel Mutation in the SCN4A Gene in a Japanese Family with Paramyotonia Congenita

Paramyotoniacongenita is an autosomal-dominant muscle disease caused by missense mutations in SCN4A, the gene enconding the alpha subunit of skeletal muscle sodium channel. It is clinically characterized by paradoxical myotonia, an attack of muscle stiffness that is aggravated by repeated activity, as well by cold-induced muscle stiffness. We describe the clinical and genetic features of a Japanese family with Paramyotoniacongenita. Five members of this family (four generations) were affected. Treatment with mexiletine, an antiarrhythmic drug that inhibits inward sodium current, relieved their symptoms. We identified a novel SCN4A mutation (c.3470T>A, p.Ile1157Asn) in the affected individuals. This mutation is located on the cytoplasmic loop connecting the transmembrane segments S4 and S5 of domain 3 of the sodium channel, the site for docking with its inactivation particle. This mutation may cause the defective inactivation of the channel. Our observation provides a new insight into the genotype-phenotype correlation in sodium channel opathies.

Tracking Voltage-Dependent Conformational Changes of the VSD in Nav with LRET

Voltage-gated sodium channels (Nav) are fundamental for the generation and the propagation of action potentials. Mammalian Nav alpha subunits are single macromolecules organized in four different domains (DI-DIV). Each is composed of 6 transmembrane segments (S1-S6) from which S1-S4 constitute the voltage sensing domain (VSD) and with S5 and S6 constituting the pore. While Nav function has been studied extensively, the exact structural mechanisms of gating are not fully understood. Recently, the crystal structure of the prokaryotic sodium channel, NavAb, has been solved, but NavAb is a homotetrameric protein in contract to the mammalian Navs. Thus many questions were not answered by the prokaryotic channel structures. To resolve the voltage dependent conformational changes of Nav, we tracked conformational changes of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4) using Lanthanide-based Resonance Energy Transfer (LRET), a FRET technique that allows for precise measurement of intermolecular distances by taking advantage of the special properties of lanthanide as an energy donor. We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which holds a lanthanide (Tb3+) ion with high affinity, inserted at the top of the S4 segment in each domain. Also, we synthesized two toxins conjugated to dyes to function as acceptors: the pore-blocking small molecule tetrodotoxin conjugated with a HiLyte fluor488 (TTX-F), and the peptide β scorpion toxin Ts1, from the Brazilian scorpion Tityus serrulatus, conjugated with Alexa488 (Ts1-Alexa488). Having several donor positions (Tb3+ ions in LBT's) and two different acceptor positions (TTX-F and Ts1-Alexa488), we calculated multiple distances in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs that remained functionally active. The results provide new insight to structure-function information in mammalian Nav channels. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Partial block by riluzole of muscle sodium channels in myotubes from amyotrophic lateral sclerosis patients.

Denervated muscles undergo fibrillations due to spontaneous activation of voltage-gated sodium (Na+) channels generating action potentials. Fibrillations also occur in patients with amyotrophic lateral sclerosis (ALS). Riluzole, the only approved drug for ALS treatment, blocks voltage-gated Na+ channels, but its effects on muscle Na+ channels and fibrillations are yet poorly characterized. Using patch-clamp technique, we studied riluzole effect on Na+ channels in cultured myotubes from ALS patients. Needle electromyography was used to study fibrillation potentials (Fibs) in ALS patients during riluzole treatment and after one week of suspension. Patients were clinically characterized in all recording sessions. In myotubes, riluzole (1 μM, a therapeutic concentration) reduced Na+ current by 20%. The rate of rise and amplitude of spikes evoked by depolarizing stimuli were also reduced. Fibs were detected in all patients tested during riluzole treatment and riluzole washout had no univocal effect. Our study indicates that, in human myotubes, riluzole partially blocks Na+ currents and affects action potentials but does not prevent firing. In line with this in vitro finding, muscle Fibs in ALS patients appear to be largely unaffected by riluzole.

Searching for the Interaction Sites of the Beta1 Subunit with the Voltage-Sensing Domains of Sodium Channels Using LRET

Mammalian voltage-gated sodium channels (Nav) are composed of two subunits: a monomeric pore-forming subunit (α-subunit), that contains four domains (DI-DIV), each composed of a voltage-sensing domain (VSD, S1-S4) and a pore domain (S5-S6) and auxiliary subunits (β-subunits) that are transmembrane proteins with type I topology: containing an extracellular amino-terminus, a single transmembrane segment and an intracellular carboxyl terminus. A large body of literature has shown that interaction between α- and β-subunits results in Nav with altered gating kinetics suggesting that β-subunits may directly interact with VSD. However, aspects of the stoichiometry, arrangement and molecular interaction between α and β subunits remain unclear. In this study, we explored the location of β1 in relation to the rat skeletal muscle sodium channel α-subunit (Nav1.4) using lanthanide-based resonance energy transfer (LRET) via two strategies. 1) Four Nav1.4 constructs were designed to encode a Tb3+ binding-tag (Nav1.4-LBT) on top of the S4 of each domain (DI-LBT, DII-LBT, DIII-LBT and DIV-LBT) as energy donor. A hexa-histidine-tag was inserted in β1 (6His-β1), which binds a Cu2+ ion to act as acceptor. 2) β1 constructs were designed to encode at an extracellular site an LBT that binds Tb3+ as energy donor while the acceptor was Alexa488 conjugated to Ts1, a β scorpion toxin which binds to DII-VSD in Nav1.4. For both experiments, we used Xenopus laevis oocytes co-expressing Nav1.4 and β1 constructs injected with the cRNA at 1:1 molar ratio. In preliminary results, DIV-LBT + 6His-β1 oocytes displayed robust energy transfer between Tb3+ and Cu2+, indicating that at least one β1-subunit is located at ∼28 A of S4-DIV. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Roles of Individual Disulfide Bridges in the Conformation and Activity of μ-Conotoxin GIIIA, a Peptide Blocker of Muscle Sodium Channels

Seven analogs of μ-conotoxin GIIIA (μ-GIIIA), a specific blocker of muscle sodium channels, were synthesized by replacing stepwise the three cystine residues with Ala. The circular dichroism spectra of the analogs suggested that the deletion of disulfide bonds gradually randamized a conformation. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the deletion of one disulfide bond reduced the potency to less than 1 % of control. Monocyclic analogs and a linear analog were almost inactive. Therefore, all three disulfide bridges are essential for stabilizing the specific conformation of μ-GIIIA to show biological activity.

Nondystrophic myotonia: Challenges and future directions

Non-dystrophic myotonias are rare diseases caused by mutations in skeletal muscle chloride and sodium ion channels with considerable phenotypic overlap between diseases. Common symptoms include muscle stiffness, transitory weakness, fatigue, and pain. Although seldom life-shortening, these myotonias cause life-time disability and affected individuals cannot perform many daily activities. A notable feature of the recessive form of chloride channelopathies is the presence of transient weakness. While there has been considerable progress in skeletal muscle channelopathies with regards to identifying biophysical abnormalities, the mechanism of transient weakness remains unclear. A recent study published in Experimental Neurology (Desaphy et al., 2013) explored this question further by comparing the biophysical properties of 3 chloride channel mutations associated with recessive myotonia congenita, with varying susceptibility to transient weakness. The authors identified a variety of functional defects in channel behavior among the 3 mutations, suggesting that this variability contributes to the differing phenotypes among chloride channelopathies. This commentary discusses nondystrophic myotonias, the results of Desaphy et al., and the treatment challenges in this rare disease.

Diagnosis and Outcome of SCN4A-Related Severe Neonatal Episodic Laryngospasm (SNEL): 2 New Cases

Mutations of SCN4A encoding the skeletal muscle sodium channel Nav 1.4 cause several types of disease, including sodium channel myotonias. The latter may be responsible for neonatal symptoms, including severe neonatal episodic laryngospasm (SNEL). Establishing the diagnosis of SCN4A-related SNEL early in the neonatal period is crucial because treatment is available that can reduce laryngospasm and improve vital and cerebral outcome. We report 2 new unrelated French patients who presented with SNEL. The first patient was initially diagnosed with laryngomalacia and underwent laryngeal surgery in the neonatal period before being diagnosed with myotonia at 14 months of age. The episodes of laryngospasm disappeared spontaneously, although occasional circumstances such as cold exposure could trigger laryngeal reactions; in addition, he developed myotonia corresponding to an adult myotonia permanens phenotype. This patient is now 24 years old and leading a normal life. The second patient was initially diagnosed with gastroesophageal reflux, then SNEL; his condition improved with carbamazepine treatment, and he is now 6 months old. The diagnostic sequence in both patients was the same: first, severe episodic apneic attacks necessitating hospitalization occurring in the first week of life; second, observation of muscle hypertrophy and peripheral hypertonia with a clear myotonic pattern on electromyogram (at 14 and 3 months of age, respectively); third, genetic testing revealing de novo SCN4A G1306E mutation. Both patients have had good therapeutic response to sodium channel blockers (carbamazepine or mexiletine).

Non-dystrophic myotonia: prospective study of objective and patient reported outcomes

Non-dystrophic myotonias are rare diseases caused by mutations in skeletal muscle chloride and sodium ion channels with considerable phenotypic overlap between diseases. Few prospective studies have evaluated the sensitivity of symptoms and signs of myotonia in a large cohort of patients. We performed a prospective observational study of 95 participants with definite or clinically suspected non-dystrophic myotonia recruited from six sites in the USA, UK and Canada between March 2006 and March 2009. We used the common infrastructure and data elements provided by the NIH-funded Rare Disease Clinical Research Network. Outcomes included a standardized symptom interview and physical exam; the Short Form-36 and the Individualized Neuromuscular Quality of Life instruments; electrophysiological short and prolonged exercise tests; manual muscle testing; and a modified get-up-and-go test. Thirty-two participants had chloride channel mutations, 34 had sodium channel mutations, nine had myotonic dystrophy type 2, one had myotonic dystrophy type 1, and 17 had no identified mutation. Phenotype comparisons were restricted to those with sodium channel mutations, chloride channel mutations, and myotonic dystrophy type 2. Muscle stiffness was the most prominent symptom overall, seen in 66.7% to 100% of participants. In comparison with chloride channel mutations, participants with sodium mutations had an earlier age of onset of stiffness (5 years versus 10 years), frequent eye closure myotonia (73.5% versus 25%), more impairment on the Individualized Neuromuscular Quality of Life summary score (20.0 versus 9.44), and paradoxical eye closure myotonia (50% versus 0%). Handgrip myotonia was seen in three-quarters of participants, with warm up of myotonia in 75% chloride channel mutations, but also 35.3% of sodium channel mutations. The short exercise test showed ≥10% decrement in the compound muscle action potential amplitude in 59.3% of chloride channel participants compared with 27.6% of sodium channel participants, which increased post-cooling to 57.6% in sodium channel mutations. In evaluation of patients with clinical and electrical myotonia, despite considerable phenotypic overlap, the presence of eye closure myotonia, paradoxical myotonia, and an increase in short exercise test sensitivity post-cooling suggest sodium channel mutations. Outcomes designed to measure stiffness or the electrophysiological correlates of stiffness may prove useful for future clinical trials, regardless of underlying mutation, and include patient-reported stiffness, bedside manoeuvres to evaluate myotonia, muscle specific quality of life instruments and short exercise testing.

A residue in the transmembrane segment 6 of domain I in insect and mammalian sodium channels regulate differential sensitivities to pyrethroid insecticides

Voltage-gated sodium channels are critical for electrical signaling in the nervous system. Pyrethroid insecticides exert their toxic action by modifying the gating of sodium channels. A valine to methionine mutation in the transmembrane segment 6 of domain I (IS6) of sodium channels from tobacco budworms (Heliothis virescens) has been shown to alter channel gating and reduce insect sodium channel sensitivity to pyrethroids. A valine to leucine substitution was subsequently reported in pyrethroid-resistant bedbug populations. Intriguingly, pyrethroid-resistant mammalian sodium channels possess an isoleucine at the corresponding position. To determine whether different substitutions at this position alter channel gating and confer pyrethroid resistance, we made valine to methionine, isoleucine or leucine substitutions at the corresponding position, V409, in a cockroach sodium channel and examined the gating properties and pyrethroid sensitivity of the three mutants in Xenopus oocytes. All three mutations reduced the channel sensitivity to three pyrethroids (permethrin, cismethrin and deltamethrin). V409M, but not V409I or V409L, caused 6–7mV depolarizing shifts in the voltage dependences of both activation and inactivation. V409M and V409L slowed channel activation kinetics and accelerated open-state deactivation kinetics, but V409I did not. Furthermore, the substitution of isoleucine with valine, but not with methionine nor leucine, at the corresponding position in a rat skeletal muscle sodium channel, rNav1.4, enhanced channel sensitivity to deltamethrin. Collectively, our study highlights an important role of residues at 409 in regulating not only sodium channel gating, but also the differential sensitivities of insect and mammalian sodium channels to pyrethroids.

S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.