Skeletal Muscle Sodium Channel Research Articles

HCN2-based biological pacing in a modified rat AV-block model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Eurostars - Heartpace 114245 Background Electronic pacemakers have several important shortcomings which are difficult to overcome due to their hardware-based design. Gene therapy-based biological pacemakers may provide a different platform which can overcome these limitations. Such biological pacemaker strategies center around overexpression of the pacemaker ion channel HCN2, either alone or with other transgenes to potentiate pacemaker performance, such as the skeletal muscle sodium channel SkM1. In order to develop clinically applicable pacemaker gene therapies further engineering is needed, which would benefit from a reliable small animal model. At present it is unknown if the rat AV block model can be used to evaluate ion channel-based biological pacing. Aim In the present study, we aim to validate a modified rat AV-block model for the evaluation of HCN2 and SKM1-based biological pacing. Methods AV-block was generated in female rats by epicardially applied needle ablation of the AV-nodal region, as previously described (N.K. ). Complete AV-block was confirmed 7 days post-ablation by loss of P-wave and QRS-complex association on the body surface ECG. Seven days post-ablation, 3 groups of animals were injected into the left ventricular apex with Ad5-TNNT2-HCN2, Ad5-TNNT2-SkM1 or saline (control). 14 days post-ablation, ECG analysis was performed during anesthesia under baseline and after i.v. administration of isoprenaline. Upon completion of functional testing, tissue samples were harvested followed by gene expression analysis using qPCR. Results Injection of Ad5-TNNT2-HCN2, Ad5-TNNT2-SkM1 or saline did not result in ectopic activation patterns in baseline conditions 7 days after injection. Administration of isoprenaline results in reversal of the QRS complexes 90% of total measurement time in Ad5-TNNT2-HCN2 injected animals. Saline and Ad5-TNNT2-SkM1 showed no change in activation pattern upon isoprenaline administration. Gene expression analysis revealed expression of HCN2 in the injection site following Ad5-TNNT2-HCN2 injection, with minimal spread to non-target regions. However, this analysis also showed Ad5-TNNT2-SkM1 did not express well in this context. Conclusion Our modified rat AV-block model, when administered with isoprenaline, recapitulated expected biological pacemaker activity of HCN2 gene delivery. However, lack of effective expression of SkM1 renders direct comparison of these relevant transgenes difficult. Nevertheless, we expect this rat AV-block model can provide a valuable tool for the evaluation of novel ion channel-based biological pacemaker strategies.

Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

New insights from modelling studies and molecular dynamics simulations of the DIS5-S6 extracellular linker of the skeletal muscle sodium channel NaV1.4.

In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DIS5-S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing - comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.

Muscle fat replacement and contractility in patients with skeletal muscle sodium channel disorders

Skeletal muscle sodium channel disorders give rise to episodic symptoms such as myotonia and/or periodic paralysis. Chronic symptoms with permanent weakness are not considered characteristic of the phenotypes. Muscle fat replacement represents irreversible damage that inevitably will impact on muscle strength. This study investigates muscle fat replacement and contractility in patients with pathogenic SCN4A variants compared to healthy controls. T1-weighted and 2-point Dixon MRI of the legs were conducted to assess fat replacement. Stationary dynamometry was used to assess muscle strength. Contractility was determined by maximal muscle contraction divided by cross-sectional muscle area. The average cross-sectional intramuscular fat fraction was greater in patients compared with controls by 2.5% in the calves (95% CI 0.74–4.29%, p = 0.007) and by 2.0% in the thighs (95% CI 0.75–3.2%, p = 0.003). Muscle contractility was less in patients vs. controls by 14–27% (p < 0.05). Despite greater fat fraction and less contractility, absolute strength was not significantly less. This study quantitatively documents greater fat fraction and additionally describes difference in muscle contractility in a large cohort of patients with skeletal muscle sodium channel disorders. The clinical impact of these abnormal findings is likely limited as muscle hypertrophy in the patients served to preserve absolute muscle strength. Subgroup analysis indicated significant difference in phenotype by genotype, however these findings lack statistical significance and serve as inspiration for future researchers to probe into the geno- phenotype relationship in these disorders.Trial registration: The study was registered at http://clinicaltrials.gov (identifier: NCT04808388).

Blockers of Skeletal Muscle Nav1.4 Channels: From Therapy of Myotonic Syndrome to Molecular Determinants of Pharmacological Action and Back.

The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure-activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activity.

Exploring the Pivotal Components Influencing the Side Effects Induced by an Analgesic-Antitumor Peptide from Scorpion Venom on Human Voltage-Gated Sodium Channels 1.4 and 1.5 through Computational Simulation.

Voltage-gated sodium channels (VGSCs, or Nav) are important determinants of action potential generation and propagation. Efforts are underway to develop medicines targeting different channel subtypes for the treatment of related channelopathies. However, a high degree of conservation across its nine subtypes could lead to the off-target adverse effects on skeletal and cardiac muscles due to acting on primary skeletal muscle sodium channel Nav1.4 and cardiac muscle sodium channel Nav1.5, respectively. For a long evolutionary process, some peptide toxins from venoms have been found to be highly potent yet selective on ion channel subtypes and, therefore, hold the promising potential to be developed into therapeutic agents. In this research, all-atom molecular dynamic methods were used to elucidate the selective mechanisms of an analgesic-antitumor β-scorpion toxin (AGAP) with human Nav1.4 and Nav1.5 in order to unravel the primary reason for the production of its adverse reactions on the skeletal and cardiac muscles. Our results suggest that the rational distribution of residues with ring structures near position 38 and positive residues in the C-terminal on AGAP are critical factors to ensure its analgesic efficacy. Moreover, the substitution for residues with benzene is beneficial to reduce its side effects.

Bioisosteric Modification of To042: Synthesis and Evaluation of Promising Use-Dependent Inhibitors of Voltage-Gated Sodium Channels.

Three analogues of To042, a tocainide‐related lead compound recently reported for the treatment of myotonia, were synthesized and evaluated in vitro as skeletal muscle sodium channel blockers possibly endowed with enhanced use‐dependent behavior. Patch‐clamp experiments on hNav1.4 expressed in HEK293 cells showed that N‐[(naphthalen‐1‐yl)methyl]‐4‐[(2,6‐dimethyl)phenoxy]butan‐2‐amine, the aryloxyalkyl bioisostere of To042, exerted a higher use‐dependent block than To042 thus being able to preferentially block the channels in over‐excited membranes while preserving healthy tissue function. It also showed the lowest active transport across BBB according to the results of P‐glycoprotein (P‐gp) interacting activity evaluation and the highest cytoprotective effect on HeLa cells. Quantum mechanical calculations and dockings gave insights on the most probable conformation of the aryloxyalkyl bioisostere of To042 in solution and the target residues involved in the binding, respectively. Both approaches indicated the conformations that might be adopted in both the unbound and bound state of the ligand. Overall, N‐[(naphthalen‐1‐yl)methyl]‐4‐[(2,6‐dimethyl)phenoxy]butan‐2‐amine exhibits an interesting toxico‐pharmacological profile and deserves further investigation.

CHANNELOPATHIES AND RELATED DISORDERS: EP.226 Muscle fat fraction, cross-sectional area and contractility in patient with skeletal muscle sodium channel disorders

We present a cross-sectional, observational study that investigates the presence of muscle degeneration in patients with skeletal muscle sodium channel disorders (SCN4A gene mutation). This includes paramyotonia congenita (PMC), hyperkalemic periodic paralysis (HyperPP) and potassium-aggravated myotonia (PAM). Little is known about quantitative fat infiltration and contractility of patients with these disorders. So far, we have studied 13 patients (30 planned), older than 18 years and diagnosed with SCN4A mutations using dynamometry testing and quantitative (Dixon) muscle MRI.

Safinamide's potential in treating nondystrophic myotonias: Inhibition of skeletal muscle voltage-gated sodium channels and skeletal muscle hyperexcitability in vitro and in vivo

The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNav1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of −120 mV, the half-maximum inhibitory concentrations (IC50) were 160 and 33 μM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 μM for closed channels and 9 μM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of −90 mV and 50-Hz stimulation, safinamide inhibited INa with an IC50 of 6 μM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC50 of 13 μM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED50 of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.

Cysteine Mutagenesis to Probe Site-Specific Interactions in the Voltage Sensor Module of hNaV1.4

Cysteine scanning mutagenesis was used to probe interactions of positively charged S4 residues with S1-S3 countercharges in domains I, II and IV of the human skeletal muscle sodium channel hNaV1.4. Single cysteine substitution was used to assess the effect of charge neutralization of residues in the voltage sensor module of these domains. Activation and fast inactivation were most affected by cysteine substitution in domains II and IV respectively, while both of these state transitions were affected by like mutations in domain I. Double cysteine mutations in the VSM of each of these domains did not result in disulfide locking. To probe for specific interactions using these mutations, we employed a metal ion bridge approach using cadmium, and assessed its effect on equilibrium parameters of activation and fast inactivation, and peak current amplitude. In domain I, double cysteine mutations of R219C or R222C with the gating charge transfer glutamate (E171C) significantly affected fast inactivation, with activation midpoint significantly depolarized by several DIS4 cysteine / countercharge mutations incorporating the polar residue substitutions N144C and S134C. Domain II double cysteine mutations R672C / E598C and R675C / E598C also significantly depolarized the activation midpoint with cadmium addition. In domain IV, double cysteine mutations R1448C / E1373C and R1448C / N1389C responded to cadmium with slowed entry into fast inactivation, a depolarized inactivation midpoint and a decreased slope. Our results show that certain domain specific functions of the voltage sensor modules in hNaV1.4 occur with pairwise interactions of S4 positive and S1-S3 countercharge residues. This work was supported by NIH R15NS093579-01A1 to JRG and NIH P20GM103408 (INBRE).

Safinamide effects on skeletal muscle sodium channels and models of non-dystrophic myotonia (NDM) compared to mexiletine: Exploring potential for clinical utility

The antiarrhythmic sodium channel blocker mexiletine is used clinically to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or poor therapeutic response. Safinamide (Xadago®) is licensed for Parkinson’s disease treatment. Besides MAO-B inhibition, safinamide modulates glutamate release through blockade of neuronal sodium channels. The in-vitro and in-vivo effects of safinamide compared to mexiletine were investigated in skeletal muscle hNav1.4 sodium channels and in rat models of myotonia. Using patch-clamp, safinamide reversibly inhibited wild-type hNav1.4 sodium currents (INa) with an IC50 of 160 and 33 μM at stimulation frequencies of 0.1 and 10 Hz respectively, compared to 256 and 46 μM for mexiletine (holding potential (hp) −120 mV). In a condition mimicking myotonia, i.e. hp of -90 mV and 50-Hz stimulation, both drugs exerted similar INa inhibition with IC50 of 5-6 μM. When applied in vitro to isolated rat skeletal muscle fibers in a myotonia-like condition induced by 9-anthracene carboxylic acid (9-AC), safinamide inhibited action potential firing with an IC50 of 13 μM, compared to 55 μM for mexiletine. In vivo in rats, oral safinamide (ED50 1.2 mg/kg) was more potent than mexiletine (ED50 7.0 mg/kg) in counteracting 9-AC induced myotonia. Safinamide inhibited myotonic skeletal muscle sodium channels in vitro and had more potent antimyotonic properties than mexiletine in vivo. Further work is warranted to evaluate if safinamide may be clinically effective for NDM.

P1599Sodium channel gene therapy with dual adeno-associated viral vectors

Abstract Background Sodium channel gene therapy carries significant potential for treatment of acquired and inherited arrhythmias. However, delivery is challenging to the length of the transgene that exceeds the packaging capacity of Adeno-Associated Virus (AAV), the most advanced long-term gene therapy vector to date. To overcome this issue, we have developed dual AAV vectors for the delivery of the skeletal muscle sodium channel 1 (SkM1). Purpose To achieve cardiac delivery of SkM1 and other large therapeutic genes using dual AAV vectors. Methods Dual AAV vectors were constructed, containing SkM1 gene fragments that allow reconstruction in the target cells by trans-splicing and recombination. An oversized single AAV vector containing SkM1 served as a control. HEK293T cells and neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with dual AAV vectors or oversized single AAV vector at an MOI of 50,000 per vector. Etoposide and Teniposide were added 2h before transduction to improve the efficiency. SkM1 mRNA and protein were isolated 3 days post transduction and the expression was detected by RT-qPCR and Western blot. Results Robust full-length SkM1 protein expression was detected in both HEK cells and NRVMs transduced by dual AAV vectors while no expression was detected in the control. Transduction with dual AAV vectors also showed significantly higher SkM1 mRNA expression in both cell types (4 to 29-fold) comparing to oversized single AAV vector. Moreover, a relatively high level of SkM1 mRNA expression was achieved in NRVM; 22-fold higher than the native cardiac sodium channel. Conclusion Efficient delivery and expression of SkM1 was successfully achieved in vitro by hybrid dual AAV vectors. This approach supports the application of SkM1 and other sodium channel antiarrhythmic gene therapies. In vivo validation and functional testing are currently in on-going.

P.99A multiplex in situ hybridisation and immunohistochemical (ISH-IHC) assay to study developmental changes in relation to fibre type and sodium channels in human skeletal muscle and their contribution to disease severity

SCN4A codes for a skeletal muscle sodium channel (SCN) that is essential for muscle excitability and contraction. SCN4A mutations cause a number of neuromuscular phenotypes including myotonia and periodic paralysis. Infants with SCN4A mutations have common and/or severe respiratory compromise include life-threatening apnoeas, whilst adults do not. Recently we described SCN4A-linked sudden infant death. Surviving infants show a spontaneous reduction in respiratory episodes with age, even if untreated, suggesting a developmental contribution to the clinical phenotype. Lack of isoform-specific SCN monoclonal antibodies are a major bottleneck in functional and pathological studies of SCN gene mutations. To study the expression of SCNs in relation to the fibre type and developmental stage we have developed two multiplex assays for testing on whole transverse frozen sections from control human skeletal muscle biopsies (0-16 years) in whom a primary neuromuscular disorder is excluded. Samples are recruited from the MRC CNMD biobank in London. Assay 1 (ISH-IHC) includes fluorescent multiplex labelling of isoform-specific mRNA probes to SCN4A, SCN5A (ACDBio), and antibodies to pan-fast/slow myosin heavy chain and laminin alpha-2, latter used as mask for myofibre identification. Assay 2 is a fully automated multi-coloured chromogenic IHC protocol (Roche Diagnostics) allowing detection of myosin heavy chain isoforms I, IIA and IIX in a single skeletal muscle section. Further assay optimisation including creating an Assay-1-2 hybrid and developing a digital script for mRNA quantification are in progress. Age-stratified data on fibre-type composition and fibre-specific SCN4A/5A-mRNA expression will be presented. Knowledge of developmental changes in fibre-type proportions may help in understanding differences in phenotype of infants and adults with SCN4A mutations. mRNA expression data will be valuable in validating future isoform-specific SCN antibodies.

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10−12 to 10−5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

NaV1.6 and NaV1.7 channels are major endogenous voltage-gated sodium channels in ND7/23 cells

ND7/23 cells are gaining traction as a host model to express peripheral sodium channels such as NaV1.8 and NaV1.9 that have been difficult to express in widely utilized heterologous cells, like CHO and HEK293. Use of ND7/23 as a model cell to characterize the properties of sodium channels requires clear understanding of the endogenous ion channels. To define the nature of the background sodium currents in ND7/23 cells, we aimed to comprehensively profile the voltage-gated sodium channel subunits by endpoint and quantitative reverse transcription-PCR and by whole-cell patch clamp electrophysiology. We found that untransfected ND7/23 cells express endogenous peak sodium currents that average –2.12nA (n = 15) and with kinetics typical of fast sodium currents having activation and inactivation completed within few milliseconds. Furthermore, sodium currents were reduced to virtually nil upon exposure to 100nM tetrodotoxin, indicating that ND7/23 cells have essentially null background for tetrodotoxin-resistant (TTX-R) currents. qRT-PCR profiling indicated a major expression of TTX-sensitive (TTX-S) NaV1.6 and NaV1.7 at similar levels and very low expression of TTX-R NaV1.9 transcripts. There was no expression of TTX-R NaV1.8 in ND7/23 cells. There was low expression of NaV1.1, NaV1.2, NaV1.3 and no expression of cardiac or skeletal muscle sodium channels. As for the sodium channel auxiliary subunits, β1 and β3 subunits were expressed, but not the β2 and β4 subunits that covalently associate with the α-subunits. In addition, our results also showed that only the mouse forms of NaV1.6, NaV1.7 and NaV1.9 sodium channels were expressed in ND7/23 cells that was originally generated as a hybridoma of rat embryonic DRG and mouse neuroblastoma cell-line. By molecular profiling of auxiliary β- and principal α-subunits of the voltage gated sodium channel complex, our results define the background sodium channels expressed in ND7/23 cells, and confirm their utility for detailed functional studies of emerging pain channelopathies ascribed to mutations of the TTX-R sodium channels of sensory neurons.

Molecular game theory for a toxin-dominant food chain model.

Animal toxins that are used to subdue prey and deter predators act as the key drivers in natural food chains and ecosystems. However, the predators of venomous animals may exploit feeding adaptation strategies to overcome toxins their prey produce. Much remains unknown about the genetic and molecular game process in the toxin-dominant food chain model. Here, we show an evolutionary strategy in different trophic levels of scorpion-eating amphibians, scorpions and insects, representing each predation relationship in habitats dominated by the paralytic toxins of scorpions. For scorpions preying on insects, we found that the scorpion α-toxins irreversibly activate the skeletal muscle sodium channel of their prey (insect, BgNaV1) through a membrane delivery mechanism and an efficient binding with the Asp/Lys-Tyr motif of BgNaV1. However, in the predatory game between frogs and scorpions, with a single point mutation (Lys to Glu) in this motif of the frog's skeletal muscle sodium channel (fNaV1.4), fNaV1.4 breaks this interaction and diminishes muscular toxicity to the frog; thus, frogs can regularly prey on scorpions without showing paralysis. Interestingly, this molecular strategy also has been employed by some other scorpion-eating amphibians, especially anurans. In contrast to these amphibians, the Asp/Lys-Tyr motifs are structurally and functionally conserved in other animals that do not prey on scorpions. Together, our findings elucidate the protein-protein interacting mechanism of a toxin-dominant predator-prey system, implying the evolutionary game theory at a molecular level.

Myasthenic congenital myopathy from recessive mutations at a single residue in NaV1.4.

ObjectiveTo identify the genetic and physiologic basis for recessive myasthenic congenital myopathy in 2 families, suggestive of a channelopathy involving the sodium channel gene, SCN4A.MethodsA combination of whole exome sequencing and targeted mutation analysis, followed by voltage-clamp studies of mutant sodium channels expressed in fibroblasts (HEK cells) and Xenopus oocytes.ResultsMissense mutations of the same residue in the skeletal muscle sodium channel, R1460 of NaV1.4, were identified in a family and a single patient of Finnish origin (p.R1460Q) and a proband in the United States (p.R1460W). Congenital hypotonia, breathing difficulties, bulbar weakness, and fatigability had recessive inheritance (homozygous p.R1460W or compound heterozygous p.R1460Q and p.R1059X), whereas carriers were either asymptomatic (p.R1460W) or had myotonia (p.R1460Q). Sodium currents conducted by mutant channels showed unusual mixed defects with both loss-of-function (reduced amplitude, hyperpolarized shift of inactivation) and gain-of-function (slower entry and faster recovery from inactivation) changes.ConclusionsNovel mutations in families with myasthenic congenital myopathy have been identified at p.R1460 of the sodium channel. Recessive inheritance, with experimentally established loss-of-function, is a consistent feature of sodium channel based myasthenia, whereas the mixed gain of function for p.R1460 may also cause susceptibility to myotonia.

Closed- and open-state models of human skeletal muscle sodium channel

Voltage-gated sodium channels play important roles in human physiology. However, their complexity hinders the understanding of their physiology and pathology at atomic level. We took advantage of the structural reports of similar channels obtained by cryo-EM (EeNav1.4, and NavPaS), and constructed models of human Nav1.4 channels at closed and open states. The open-state model is very similar to the recently published cryo-EM structure of hNav1.4. The comparison of both models shows shifts of the voltage sensors (VS) of DIII and DIV. The activated position of VS-DII in the closed model was demonstrated by Ts1 docking, thereby confirming the requirement that VS-DI, VS-DII and VS-DIII must be activated for the channel to open. The interactions observed with VS-DIII suggest a stepwise, yet fast, transition from resting to activated state. These models provide structural insights on the closed-open transition of the channel.

NMR Structure of μ-Conotoxin GIIIC: Leucine 18 Induces Local Repacking of the N-Terminus Resulting in Reduced NaV Channel Potency.

μ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of μ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of μ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical μ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (NaV), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.

Large-effect mutations generate trade-off between predatory and locomotor ability during arms race coevolution with deadly prey.

Adaptive evolution in response to one selective challenge may disrupt other important aspects of performance. Such evolutionary trade‐offs are predicted to arise in the process of local adaptation, but it is unclear if these phenotypic compromises result from the antagonistic effects of simple amino acid substitutions. We tested for trade‐offs associated with beneficial mutations that confer tetrodotoxin (TTX) resistance in the voltage‐gated sodium channel (NaV1.4) in skeletal muscle of the common garter snake (Thamnophis sirtalis). Separate lineages in California and the Pacific Northwest independently evolved TTX‐resistant changes to the pore of NaV1.4 as a result of arms race coevolution with toxic prey, newts of the genus Taricha. Snakes from the California lineage that were homozygous for an allele known to confer large increases in toxin resistance (NaV1.4LVNV) had significantly reduced crawl speed compared to individuals with the ancestral TTX‐sensitive channel. Heterologous expression of native snake NaV1.4 proteins demonstrated that the same NaV1.4LVNV allele confers a dramatic increase in TTX resistance and a correlated decrease in overall channel excitability. Our results suggest the same mutations that accumulate during arms race coevolution and beneficially interfere with toxin‐binding also cause changes in electrophysiological function of the channel that may affect organismal performance. This trade‐off was only evident in the predator lineage where coevolution has led to the most extreme resistance phenotype, determined by four critical amino acid substitutions. If these biophysical changes also translate to a fitness cost—for example, through the inability of T. sirtalis to quickly escape predators—then pleiotropy at this single locus could contribute to observed variation in levels of TTX resistance across the mosaic landscape of coevolution.