NaV1.4 is a voltage-gated sodium channel subtype that is predominantly expressed in skeletal muscle cells. It is essential for producing action potentials and stimulating muscle contraction, and mutations in NaV1.4 can cause various muscle disorders. The discovery of the cryo-EM structure of NaV1.4 in complex with β1 has opened new possibilities for designing drugs and toxins that target NaV1.4. In this review, we summarize the current understanding of channelopathies, the binding sites and functions of chemicals including medicine and toxins that interact with NaV1.4. These substances could be considered novel candidate compounds or tools to develop more potent and selective drugs targeting NaV1.4. Therefore, studying NaV1.4 pharmacology is both theoretically and practically meaningful.

Skeletal muscle sodium channelopathies due to SCN4A gene mutations have a broad clinical spectrum. However, each phenotype has been reported in few cases of Chinese origin. We present detailed phenotype and genotype data from a cohort of 40 cases with SCN4A gene mutations seen in neuromuscular diagnostic service in Huashan hospital, Fudan University. Cases were referred from 6 independent provinces from 2010 to 2018. A questionnaire covering demographics, precipitating factors, episodes of paralysis and myotonia was designed to collect the clinical information. Electrodiagnostic studies and muscle MRI were retrospectively analyzed. The clinical spectrum of patients included: 6 Hyperkalemic periodic paralysis (15%), 18 Hypokalemic periodic paralysis (45%), 7 sodium channel myotonia (17.5%), 4 paramyotonia congenita (10%) and 5 heterozygous asymptomatic mutation carriers (12.5%). Review of clinical information highlights a significant delay to diagnosis (median 15 years), reports of pain and myalgia in the majority of patients, male predominance, circadian rhythm and common precipitating factors. Electrodiagnostic studies revealed subclinical myotonic discharges and a positive long exercise test in asymptomatic carriers. Muscle MRI identified edema and fatty infiltration in gastrocnemius and soleus. A total of 13 reported and 2 novel SCN4A mutations were identified with most variants distributed in the transmembrane helix S4 to S6, with a hotspot mutation p.Arg675Gln accounting for 32.5% (13/40) of the cohort. Our study revealed a higher proportion of periodic paralysis in SCN4A-mutated patients compared with cohorts from England and the Netherlands. It also highlights the importance of electrodiagnostic studies in diagnosis and segregation studies.

Reciprocal adaptation is the hallmark of arms race coevolution. Local coadaptation between natural enemies should generate a geographic mosaic pattern where both species have roughly matched abilities across their shared range. However, mosaic variation in ecologically relevant traits can also arise from processes unrelated to reciprocal selection, such as population structure or local environmental conditions. We tested whether these alternative processes can account for trait variation in the geographic mosaic of arms race coevolution between resistant garter snakes (Thamnophis sirtalis) and toxic newts (Taricha granulosa). We found that predator resistance and prey toxin levels are functionally matched in co-occurring populations, suggesting that mosaic variation in the armaments of both species results from the local pressures of reciprocal selection. By the same token, phenotypic and genetic variation in snake resistance deviates from neutral expectations of population genetic differentiation, showing a clear signature of adaptation to local toxin levels in newts. Contrastingly, newt toxin levels are best predicted by genetic differentiation among newt populations, and to a lesser extent, by the local environment and snake resistance. Exaggerated armaments suggest that coevolution occurs in certain hotspots, but prey population structure seems to be of particular influence on local phenotypic variation in both species throughout the geographic mosaic. Our results imply that processes other than reciprocal selection, like historical biogeography and environmental pressures, represent an important source of variation in the geographic mosaic of coevolution. Such a pattern supports the role of \"trait remixing\" in the geographic mosaic theory, the process by which non-adaptive forces dictate spatial variation in the interactions among species.

Myotonia congenita (MC) is a rare disorder characterized by stiffness and weakness of the limb and trunk muscles. Mutations in the SCN4A gene encoding the alpha-subunit of the voltage-gated sodium channel Nav1.4 have been reported to be responsible for sodium channel myotonia (SCM). The Nav1.4 channel is expressed in skeletal muscles, and its related channelopathies affect skeletal muscle excitability, which can manifest as SCM, paramyotonia and periodic paralysis. In this study, the missense mutation p.V445M was identified in two individual families with MC. To determine the functional consequences of having a mutated Nav1.4 channel, whole-cell patch-clamp recording of transfected Chinese hamster ovary cells was performed. Evaluation of the transient Na+ current found that a hyperpolarizing shift occurs at both the activation and inactivation curves with an increase of the window currents in the mutant channels. The Nav1.4 channel\'s co-expression with the Navβ4 peptide can generate resurgent Na+ currents at repolarization following a depolarization. The magnitude of the resurgent currents is higher in the mutant than in the wild-type (WT) channel. Although the decay kinetics are comparable between the mutant and WT channels, the time to the peak of resurgent Na+ currents in the mutant channel is significantly protracted compared with that in the WT channel. These findings suggest that the p.V445M mutation in the Nav1.4 channel results in an increase of both sustained and resurgent Na+ currents, which may contribute to hyperexcitability with repetitive firing and is likely to facilitate recurrent myotonia in SCM patients.

[This corrects the article DOI: 10.3389/fneur.2020.00077.].

Mutations of the voltage-gated sodium channel gene (SCN4A), which encodes Nav1.4, cause nondystrophic myotonia that occasionally is associated with severe apnea and laryngospasm. There are case reports of nondystrophic myotonia due to mutations in the C-terminal tail (CTerm) of Nav1.4, but the functional analysis is scarce.
We present two families with nondystrophic myotonia harboring a novel heterozygous mutation (E1702del) and a known heterozygous mutation (E1702K).
The proband with E1702K exhibited repeated rhabdomyolysis, and the daughter showed laryngospasm and cyanosis. Functional analysis of the two mutations as well as another known heterozygous mutation (T1700_E1703del), all located on EF hand-like motif in CTerm, was conducted with whole-cell recording of heterologously expressed channel. All mutations displayed impaired fast inactivation.
The CTerm of Nav1.4 is vital for regulating fast inactivation. The study highlights the importance of accumulating pathological mutations of Nav1.4 and their functional analysis data.

The phenotypic spectrum associated with the skeletal muscle voltage-gated sodium channel gene (SCN4A) has expanded with advancements in genetic testing. Autosomal dominant SCN4A mutations were first linked to hyperkalemic periodic paralysis, then subsequently included paramyotonia congenita, several variants of myotonia, and finally hypokalemic periodic paralysis. Biallelic recessive mutations were later identified in myasthenic myopathy and in infants showing a severe congenital myopathy with hypotonia. We report a patient with a pathogenic de novo SCN4A variant, c.2386C>G p.L769V at a highly conserved leucine. The phenotype was manifest at birth with arthrogryposis multiplex congenita, severe episodes of bronchospasm that responded immediately to carbamazepine therapy, and electromyographic evidence of widespread myotonia. Another de novo case of p.L769V has been reported with hip dysplasia, scoliosis, myopathy, and later paramyotonia. Expression studies of L796V mutant channels showed predominantly gain-of-function changes, that included defects of slow inactivation. Computer simulations of muscle excitability reveal a strong predisposition to myotonia with exceptionally prolonged bursts of discharges, when the L796V defects are included. We propose L769V is a pathogenic variant, that along with other cases in the literature, defines a new dominant SCN4A disorder of myotonic myopathy with secondary congenital joint and skeletal involvement.

The mechanism of inactivation of mammalian voltage-gated Na+ channels involves transient interactions between intracellular domains resulting in direct pore occlusion by the IFM motif and concomitant extracellular interactions with the β1 subunit. Navβ1 subunits constitute single-pass transmembrane proteins that form protein-protein associations with pore-forming α subunits to allosterically modulate the Na+ influx into the cell during the action potential of every excitable cell in vertebrates. Here, we explored the role of the intracellular IFM motif of rNav1.4 (skeletal muscle isoform of the rat Na+ channel) on the α-β1 functional interaction and showed for the first time that the modulation of β1 is independent of the IFM motif. We found that: (1) Nav1.4 channels that lack the IFM inactivation particle can undergo a \"C-type-like inactivation\" albeit in an ultraslow gating mode; (2) β1 can significantly accelerate the inactivation of Nav1.4 channels in the absence of the IFM motif. Previously, we identified two residues (T109 and N110) on the β1 subunit that disrupt the α-β1 allosteric modulation. We further characterized the electrophysiological effects of the double alanine substitution of these residues demonstrating that it decelerates inactivation and recovery from inactivation, abolishes the modulation of steady-state inactivation and induces a current rundown upon repetitive stimulation, thus causing a general loss of function. Our results contribute to delineating the process of the mammalian Na+ channel inactivation. These findings may be relevant to the design of pharmacological strategies, targeting β subunits to treat pathologies associated to Na+ current dysfunction.

Hyperkalemic periodic paralysis (HyperPP) is a dominantly inherited muscle disease caused by mutations in SCN4A gene encoding skeletal muscle voltage gated Nav 1.4 channels. We identified a novel Nav 1.4 mutation I692M in 14 families out of the 104 genetically identified HyperPP families in the Neuromuscular Centre Ulm and is therefore as frequent as I693T (13 families out of 14 HyperPP families) in Germany. Surprisingly, in 13 families, a known polymorphism S906T was also present. It was on the affected allele in at least 10 families compatible with a possible founder effect in central Europe. All affected members suffered from episodic weakness; myotonia was also common. Compared with I692M patients, I692M-S906T patients had longer weakness episodes, more affected muscles, CK elevation and presence of permanent weakness. Electrophysiological investigation showed that both mutants had incomplete slow inactivation and a hyperpolarizing shift of activation which contribute to membrane depolarization and weakness. Additionally, I692M-S906T significantly enhanced close-state fast inactivation compared with I692M alone, suggesting a higher proportion of inactivated I692M-S906T channels upon membrane depolarization which may facilitate the initiation of weakness episodes and therefore clinical manifestation. Our results suggest that polymorphism S906T has effects on the clinical phenotypic and electrophysiological severity of a novel borderline Nav 1.4 mutation I692M, making the borderline mutation fully penetrant.

Mutations in Nav1.4 and Nav1.5 α-subunits have been associated with muscular and cardiac channelopathies, respectively. Despite intense research on the structure and function of these channels, a lot of information is still missing to delineate the various physiological and pathophysiological processes underlying their activity at the molecular level. Nav1.4 and Nav1.5 sequences are similar, suggesting structural and functional homologies between the two orthologous channels. This also suggests that any characteristics described for one channel subunit may shed light on the properties of the counterpart channel subunit. In this review article, after a brief clinical description of the muscular and cardiac channelopathies related to Nav1.4 and Nav1.5 mutations, respectively, we compare the knowledge accumulated in different aspects of the expression and function of Nav1.4 and Nav1.5 α-subunits: the regulation of the two encoding genes (SCN4A and SCN5A), the associated/regulatory proteins and at last, the functional effect of the same missense mutations detected in Nav1.4 and Nav1.5. First, it appears that more is known on Nav1.5 expression and accessory proteins. Because of the high homologies of Nav1.5 binding sites and equivalent Nav1.4 sites, Nav1.5-related results may guide future investigations on Nav1.4. Second, the analysis of the same missense mutations in Nav1.4 and Nav1.5 revealed intriguing similarities regarding their effects on membrane excitability and alteration in channel biophysics. We believe that such comparison may bring new cues to the physiopathology of cardiac and muscular diseases.