Na_v channels are composed of one large, pore-forming α subunit with one or two auxiliary β subunits. Nine Na_v isoforms (Na_v1.1-Na_v1.9) are described in mammals. Each isoform is characterized by its electrophysiological and pharmacological properties, as well as its tissue expression pattern. An additional and atypical Na⁺ channel isoform, Na_x, is not voltage-dependent and arises as a different Na⁺ channel subfamily (Dolivo et al., 2021). The pore-forming α subunit of Na_v forms a functional Na⁺ channel and the β subunits modulate its trafficking and electrophysiological properties. The α subunit is organized in four homologous transmembrane repeat domains (DI-DIV). Each domain contains six transmembrane segments (S1-S6) that are divided into two main functional modules: the voltage-sensing domain (S1-S4) and the pore module (S5-S6) (Figure 2). The β subunits are multifunctional glycoproteins with one single transmembrane segment, and four isoforms exist in mammals (β1-β4 encoded by SCN1B-SCN4B) (Winters and Isom, 2016).

Na_v channels have three main distinct conformational states: resting (closed), activated (open), inactivated (closed). The kinetics of each transition, i.e. activation (from resting to activated), inactivation (from activated to inactivated) and recovery from inactivation (from inactivated to resting) shape the electrophysiological behavior of Na_v channels and critically determine AP frequency. Inactivation includes two distinct components: fast-inactivation and slow-inactivation, fast-inactivation being the main feature of Na_v channels. Na_v channels activate rapidly (in less than 1 ms) in response to membrane depolarization. The major structural determinants in this process are the S4 transmembrane segments. They are composed of positively charged arginine (or lysine) residues, also called gating charges, occurring in a Arg/Lys-X-X repetitive sequence (Figure 2). Upon depolarization, the S4 helices undergo outward movements by electrostatic interactions of their basic residues with conserved acidic or polar residues of S2 and S3 segments (Catterall et al., 2020). This outward movement drives the conformational shifts of the channel in response to depolarization with opening of the central (also known as α) pore formed by the P-loops of the four S5–S6 segments. The open central pore then conducts a large inward Na⁺ current that drives the depolarization phase of AP. Within a few milliseconds following their activation, Na_v channels undergo fast inactivation, which facilitates membrane repolarization. Fast inactivation is mainly dictated by the movement of DIVS4 during activation (Capes et al., 2013). During this conformational change, the inactivation gate formed by the cytoplasmic linker between DIII and DIV intracellularly blocks the central pore. Prolonged or high-frequency depolarizations drive Na_v channels into a slow-inactivated state with time constant ranging from 100 ms to several minutes. Slow inactivation determines long-lasting Na_v channels availability, thereby defining the cell firing properties. Its structural basis remains unclear. It probably relies on several sequential conformational changes including constriction of the pore by rearrangement of the ion selectivity filter and S6 segments (Mantegazza et al., 2021).

The force developed by a muscle from a single twitch to its maximal sustained contraction (known as tetanus) resulting from the twitch summation relies on the AP frequency, and by this way on the functional properties of Na_v1.4. In the adult myofiber, the AP is generated at the neuromuscular junction (NMJ) located in the middle of the myofiber when the endplate potential (epp), a local depolarization induced by the opening of the post-synaptic nicotinic acetylcholine receptors (nAChRs), reaches the threshold (-50 mV) for AP genesis. To decrease the effective threshold for AP generation and favor the latter, the amplitude of Na_v currents is higher at the NMJ than in the extrasynaptic area (80 mA/cm² against 20 mA/cm², adult fast-twitch myofibers of mouse Levator auris longus) (Figure 3) (Lupa et al., 1993; Slater, 2003). The high synaptic Na⁺ current density results from the clustering of Na_v channels at the NMJ, more specifically in the depth of the postsynaptic folds while the tops of the folds are enriched in AChRs (Figure 3). The AP generated at the NMJ then propagates longitudinally toward the extremities and radially through the transverse tubules to drive excitation-contraction coupling and myofiber contraction. In the extrasynaptic area, the distribution of Na_v channels between the “surface” and transverse tubules sarcolemma is estimated to be in the range between 40:60 and 60:40 (DiFranco and Vergara, 2011).

Two Na_v currents exist in skeletal myofibers. In innervated adult myofibers of rodents, a tetrodotoxin (TTX)-sensitive Na⁺ current (IC₅₀ 5 nM) generated by Na_v1.4 (formerly SkM1) accounts for 98% of the whole inward Na⁺ current (Fu et al., 2011). In immature neonatal myofibers, this current is low or null compared to a TTX-resistant Na⁺ current (SkM2, IC₅₀ 2000 nM). The TTX-resistant current is mediated by Na_v1.5 encoded by SCN5A, which is the main cardiac Na_v isoform (Loussouarn et al., 2015). In addition to distinct TTX sensitivity, these two Na⁺ currents have different electrophysiological properties with higher activation time constants and lower conductance for Na_v1.5 current compared to Na_v1.4 current (Morel et al., 2010). The switch between Na_v1.5 and Na_v1.4 occurs between birth and the third postnatal week in rodents and is concomitant to the maturation of NMJs and myofibers (Lupa et al., 1993). The neonatal lethality of children and mice with bi-allelic null mutations in SCN4A demonstrates that Na_v1.5 does not compensate for the lack of Na_v1.4 in skeletal muscles at birth (Wu et al., 2016; Zaharieva et al., 2016).

Not all myofibers are equal regarding excitability and Na_v current properties. Slow-twitch motor units in mammals are active at 10–20 Hz and fast-twitch motor units are active at rates from 40 to 90 Hz to more than 200 Hz for brief periods (Hennig and Lømo, 1985). To assume these high frequencies, the Na⁺ current density is two-to six-fold higher in fast-than in slow-twitch myofibers (Ruff, 1996; Desaphy et al., 2001). The difference in synaptic versus extrasynaptic Na⁺ current density is also more important in fast-twitch compared to slow-twitch myofibers (Milton et al., 1992). Slow inactivation of Na⁺ current is less prominent in slow-twitch fibers (Ruff, 1996). These distinct characteristics probably play a role in the excitability and contractility properties of myofibers such as threshold potential, time to reach contraction threshold or resistance to fatigue.

It is here important to note inter-species differences. For example, the time constants for entry or recovery from slow inactivation are reported to be 4 times faster in human than in rat myofibers (Ruff, 1996). Individual human muscles tend to be a mixture of various fiber types. Whether and when the Na_v1.5/Na_v1.4 switch occurs in humans is not documented. Human NMJs are smaller and have more extensive postsynaptic folds than rodent NMJs, which may have functional significance since the NMJ size and the spacing and depth of the postsynaptic folds have an important effect on AP genesis (Jones et al., 2017; Slater, 2017). Moreover, proteomics analyses have recently provided evidence that human NMJs have a significantly modified molecular composition compared to mouse NMJs (Jones et al., 2017). These differences must be taken into consideration when interpreting animal-based studies with respect to their applicability to humans.

Protein interactions of Na_v channels modulate their trafficking, cellular localization and functional properties. Little is known about these important functional aspects for Na_v1.4 compared to other Na_v channels (Loussouarn et al., 2015; Eshed-Eisenbach and Peles, 2020). If sequence similarities suggest the possibility that Na_v1.4 shares binding partners and regulatory mechanisms with other Na_v isoforms, further investigations are required to prove this assumption and to best understand the fine-tuning of Na_v1.4 in skeletal myofibers.

One regulatory β subunit, mainly β1 (encoded by SCN1B), associates non-covalently with the Na_v1.4 α subunit (Yang et al., 1993). The β2 (SCN2B), β3 (SCN3B) or β4 (SCN4B) subunits might also contribute to the Na_v1.4 channel complex since their transcripts are detected in muscle cells (David et al., 2008; Morel et al., 2010). Two extracellular binding sites (DIS5S6 and DIVS5S6) of Na_v1.4 are involved in the interaction with β1 (Figure 2) (Makita et al., 1996). Different effects of the interaction of αNa_v1.4 with β1 on Na⁺ current have been reported depending on the heterologous cell expression system. They include increased current density, enhanced inactivation with faster kinetics and hyperpolarization shift (Cannon et al., 1993). The differences between cell expression systems may result from distinct β1 glycosylation, which would modify Na_v channel gating through the density of the surrounding surface charges (Ferrera and Moran, 2006). No mutations in the genes encoding β subunits are reported to cause disorders with altered skeletal muscle force (Winters and Isom, 2016). It remains to be determined whether this is due to a lower sensitivity of Na_v1.4 to functional modulation by β subunits in myofibers compared to other Na_v isoforms, to compensation between β isoforms or to the subjective nature of mild variations in muscle force, which may not be considered as “pathogenic” by individuals suffering from them.

The mechanisms leading to the synaptic accumulation of Na_v1.4 are still unclear compared to those resulting in the clustering of neuronal channels at axonal initial segments or nodes of Ranvier (Eshed-Eisenbach and Peles, 2020). Synaptic aggregation of Na_v1.4 channels would result from their binding interaction with ankyrins (Figure 3). Ankyrins are scaffolding proteins that link membrane-bound proteins to the underlying spectrin-actin cytoskeleton. Ankyrins have a pivotal role in the anchorage of Na_v channels to the submembranous cytoskeleton through their binding interaction with a motif of 27 amino acid residues located within the cytoplasmic DII-DIII linker of Na_v channels (Figure 2) (Garrido et al., 2003). A recent work has confirmed that ankyrins are required to cluster Na_v channels in the depth of synaptic folds since the lack of ankyrins G, R and B induced loss of Na_v staining at the NMJ (Zhang et al., 2021). The clustering of Na_v channels in the depth of the folds is supposed to result from the physical exclusion of the ankyrin-Na_v1.4 complexes from the tops rich in nAChRs during NMJ formation maturation (Flucher and Daniels, 1989; Bailey et al., 2003).

Na_v1.4 also interacts with α syntrophin, a peripheral membrane protein localized to the cytosolic face that links a variety of signaling proteins and ion channels to the dystrophin-associated protein complex (Figure 3) (Gee et al., 1998). The C-terminal tail of Na_v1.4 possesses a SerLeuVal peptidic sequence that binds the PDZ (for « PSD-95, Dlg1, Zo-1 ») domain of syntrophin (Figure 2). The synaptic immunostaining of syntrophin is distinct from Na_v immunostaining at the NMJ, suggesting that their interaction solely takes place in the extrasynaptic region (Bailey et al., 2003). The syntrophin-Na_v1.4 interaction could modify the gating properties of Na_v1.4 and participate to loss of muscle force in some acquired or inherited myopathic conditions, such as critical illness myopathy or Duchenne muscular dystrophy, a disease acquired by patients in intensive care units and an inherited disorder characterized by progressive muscle degeneration and weakness due to the lack of dystrophin, respectively (Teener and Rich, 2006; Hirn et al., 2008; Kraner et al., 2012).

Calmodulin is one of the rare proteins well known to modulate Na_v1.4 gating (Nathan et al., 2021). This intracellular multifunctional protein is ubiquitously expressed and acts as part of a Ca²⁺ signal transduction pathway by modifying its interactions when bound to Ca²⁺. Calmodulin interacts with an IQ domain downstream of an EF-hand domain located in the cytoplasmic C-terminal tail of Na_v1.4 (Figure 2). The interaction of Na_v1.4 with calmodulin facilitates its cell surface expression and mediates its Ca²⁺-dependent slow inactivation (Biswas et al., 2008; Yoder et al., 2019). The crystal structure of Ca²⁺-calmodulin bound to Na_v1.4 suggests that the Ca²⁺-dependent inactivation of Na_v1.4 would result from the relative reorientation of its EF-hand and IQ domains (Gardill et al., 2019). Increasing the intracellular pool of Ca²⁺ released from the sarcoplasmic reticulum inhibits Na⁺ currents in mouse skeletal muscles (Sarbjit-Singh et al., 2020; Liu et al., 2021). These data suggest activity-dependent feedback mechanisms of Na_v1.4 activity by intracellular Ca²⁺ released from the transverse tubules for a fine tuning of muscle contraction during periods of repetitive activity.

To complete this list of Na_v1.4 binding interaction, it is worth noting that the α subunits of Na_v1.5, Na_v1.1, Na_v1.2 and Na_v1.7 form functional dimers (Clatot et al., 2017; Rühlmann et al., 2020). The dimerization involves a 20 amino acid motif within the DI-DII cytoplasmic loop and is mediated by the 14-3-3 protein. Na_v1.4 is the sole Na_v channel lacking this motif, suggesting that functional dimerization does not occur for this isoform (Rühlmann et al., 2020).

The human SCN4A gene (24 exons, chromosome 17q23.3) codes for a single mRNA transcript transcribed into a protein of 1,836 amino acids (208 kDa). SCN4A is referred to as the skeletal muscle isoform since its cDNA cloning 30 years ago (Trimmer et al., 1989). A small expression of SCN4A in heart tissue and cardiac myocytes is reported (1.1% of relative Na_v channels transcript levels in humans) (Blechschmidt et al., 2008; Kaufmann et al., 2013). The tissue-specific expression of SCN4A most probably explains why Na_v1.4 dysfunction only impacts skeletal muscles even if anecdotic reports of cardiac arrhythmia in individuals with dominant SCN4A mutations have been published (Loussouarn et al., 2015).

In rodents, Scn4a expression is developmentally regulated. The amount of Scn4a transcripts increases from birth to 2 weeks after birth while Scn5a (the gene encoding the pore-forming subunit of Na_v1.5) expression decreases (Lupa et al., 1993). This developmental period is concomitant to the maturation of the neuromuscular system, especially the NMJ, in rodents (Li et al., 2018). The high density of Na_v channels at the NMJ would result in part from the accumulation of Scn4a mRNA at the synapse (Lupa et al., 1993; Awad et al., 2001; Bailey et al., 2003). However, Scn4a expression is insensitive to denervation in the adult myofiber, suggesting that its synaptic expression is not strictly regulated by the nerve terminal (Lupa and Caldwell, 1991; Awad et al., 2001; Carreras et al., 2021). Northern blot analyses have showed that Scn1b expression parallels Scn4a expression during postnatal development and after denervation in rodents, suggesting that common factors regulate their gene expression (Yang et al., 1993). Whether similar modulations of SCN4A and SCN1B gene expression with NMJ development and denervation exist in humans has not been studied.

The specific expression of Scn4a in differentiated myofibers is controlled by at least 4 cis-regulatory promotor elements working together: 1) a core promoter that lacks muscle specificity; 2) a repressor that confers muscle specificity; 3) a muscle-specific positive element, whose accessibility is partially masked by the repressor; and 4) another muscle-specific positive element that confers a 10-fold up-regulation of expression (Hebert et al., 2007; Kraner et al., 1999; Kraner et al., 1998). The cumulative binding of non-specific (Nuclear Factors (NF) I, ZEB/AREB6 and REB) and muscle-specific (myogenin and MRF4) transcription factors on these elements would contribute to the upregulation of Scn4a expression in myofibers. According to the fact that denervation does not modify its expression, no N-box—a response element that promotes synaptic-specific expression in response to the neuronal factors agrin and neuregulins for NMJ formation and maturation (Belotti and Schaeffer, 2020) — is present within the promotor of Scn4a, suggesting the involvement of other regulatory mechanisms. The simplest hypothesis is a higher synaptic amount of Scn4a mRNA due to the high number of myonuclei in this area.

Several transcripts exist for SCN5A but none is specific to skeletal myofibers. Na_v1.5 GoF or LoF due to SCN5A mutations cause cardiac arrhythmia (Long QT syndrome (LQTS), type 3 and Brugada syndrome, respectively) but are not reported to cause neuromuscular symptoms. In contrast to Scn4a, Scn5a expression is sensitive to the innervation pattern of the myofibers with downregulation during postnatal development and strong upregulation when the adult myofiber is denervated (Awad et al., 2001; Carreras et al., 2021). The upregulation of Scn5a upon denervation results from the binding of the Gata4 transcription factor, itself upregulated following denervation, to the Scn5a promoter region, and from epigenetic regulations mediated by the H3K27ac and H3K4me3 histones (Carreras et al., 2021).

Familial hypokalemic periodic paralysis (HypoPP) is a rare condition characterized by the episodic occurrence of moderate to severe muscle weakness, which may be focal or generalized, concomitantly to low blood K⁺ levels (<3,5 mEq/L). The attacks are usually triggered by rest after strenuous exercise, carbohydrate-rich meal, or stress. Each attack has a gradual onset over minutes and resolves spontaneously after few hours or days (Statland et al., 2018). Familial and non-familial forms of HypoPP exist. Non-familial forms are most often secondary to another physiological dysfunction such as thyrotoxicosis, hyperaldosteronism or nephropathic K⁺ loss. Familial forms are dominantly-inherited and typically develop in the first or second decade of life. Inter-critical electromyographic testing based on compound muscle APs (CMAP) recording demonstrates a decrease of CMAP amplitudes in response to a 5 min-long exercise, which may help to document muscle weakness between episodes (McManis et al., 1986; Fournier et al., 2004). The definitive diagnosis of HypoPP lies on genetic testing with mutations in the genes known to cause HypoPP: CACNA1S, which encodes the pore-forming subunit of the skeletal muscle voltage-gated calcium channel Ca_v1.1 (HypoPP1), or SCN4A (HypoPP2) (Jurkat-Rott et al., 1994; Ptáček et al., 1994; Jurkat-Rott et al., 2000). Dominant-negative mutations in the KCNJ2 gene, encoding the K⁺ channel Kir2.1, also cause primary HypoPP in Andersen-Tawil syndrome, a disorder clinically distinct from HypoPP1 and 2 since the neuromuscular phenotype is associated with cardiac arrhythmia and bone deformities (Plaster et al., 2001).

Fourteen SCN4A missense mutations are reported to cause HypoPP2. Eleven substitute the two arginine residues (R1 or R2) nearest the extracellular end of S4 segments in DI, DII and DIII and cause HypoPP2 that are dominantly-inherited (Figure 2 and Table 1). The exception are p.Arg1135His/Cys and p.Arg1451Leu. The p.Arg1135His/Cys mutations substitute the 3rd arginine residue of DIIIS4 and cause de novo (p.Arg1135His) or recessively-inherited (p.Arg1135Cys) HypoPP2 (Groome et al., 2014). P.Arg1451Leu substitutes the 1st arginine residue of DIVS4 and results in HypoPP2 and myotonia in one homozygous individual (Groome et al., 2014; Luo et al., 2018). HypoPP2 mutations that substitute R1 or R2 as well as p.Arg1135His/Cys, but not p.Arg1451Leu, promote a gating pore current (Sokolov et al., 2007; Jurkat-Rott et al., 2009; Wu et al., 2012). The gating pore current (also named omega current) is a cation-selective inward current activated by hyperpolarization through an aqueous pathway created by the neutralization of one of the two outermost positive charges of DIS4, DIIS4 or DIIIS4 in Na_v1.4 (Gosselin-Badaroudine et al., 2012). This small inward current explains the paradoxical depolarization characteristics of HypoPP myofibers: they are more frequently depolarized than control myofibers (resting membrane potential (RMP) equal to −55 mV instead of −90 mV) in low extracellular K⁺ (Rüdel et al., 1984). To simplify complex pathophysiological mechanisms, hypokalemia would be induced by muscle K⁺ reuptake after exercise or in response to insulin or glucocorticoid (such as cortisol) secretion following carbohydrate-rich meals or stress, respectively. Hypokalemia would modify the cumulated activities of K⁺ (especially Kir2.1) and Cl^- channels, ATPase pumps and Na-K-2Cl (NKCC) transporters in myofibers (Jurkat-Rott et al., 2009; Wu et al., 2011; Mi et al., 2019). These changes would establish the net balance of no ionic current at the depolarized (−55 mV) value in HypoPP2 myofibers because of the gating pore current. Na_v1.4 channels would then be mostly inactivated, and the myofibers not excitable (Rüdel et al., 1984; Struyk and Cannon, 2008; Jurkat-Rott et al., 2009). The HypoPP2 mutations could therefore be considered as dominant-negative, the gating pore current altering the central pore current of mutant and wild-type (WT) Na_v1.4 channels.

HypoPP2 mutations causing gating pore current have also been reported to cause LoF effects on Na_v1.4 gating in heterologous cell expression systems (Bayless-Edwards et al., 2018; Bendahhou et al., 2001; Groome et al., 2014; Jurkat-Rott et al., 2000; Mi et al., 2014; Struyk et al., 2000). The LoF changes include reduced Na⁺ current density and enhanced fast and slow inactivation (Table 1). Whether they participate to the loss of muscle force in HypoPP2 is an open question. One argument for the physiological significance of these LoF changes is the smaller amplitude and the slower rate of rise of muscle APs in HypoPP2 myofibers compared to control myofibers at normal (−90 mV) RMP (Jurkat-Rott et al., 2000; Groome et al., 2014; Bayless-Edwards et al., 2018). Moreover, introducing one HypoPP2 missense mutation (p.Arg669His) into the mouse Scn4a gene by homologous recombination reproduces LoF changes on muscle APs in addition to the pathogenic gating pore current (Wu et al., 2011). A second argument comes from recent reports of SCN4A missense mutations that do not induce gating pore current in individuals suffering from PP with hypokalemic episodes of muscle weakness (Kokunai et al., 2018; Luo et al., 2018; Poulin et al., 2018). Two individuals are heterozygous for one missense substitution (p.Ala204Glu, p.Arg1451Leu) whereas one is homozygous for one of these two substitutions (p.Arg1451Leu). The recurrence of p.Arg1451Leu in unrelated individuals underlines the link between the functional changes induced by this SCN4A mutation and hypokalemic episodes of paralysis. P.Ala204Glu and p.Arg1451Leu exert GoF effects with enhanced activation. They also exert LoF effects with reduced Na⁺ current density, slower activation kinetics, accelerated entry into fast inactivation and slower recovery from slow inactivation for mutant channels compared to WT channels (Kokunai et al., 2018; Luo et al., 2018). For one (p.Ala204Glu), LoF changes were more severe in low K⁺ _ext with a depolarization shift of activation observed for the mutant but not the WT channels (Kokunai et al., 2018). Recently, a charge-retaining substitution in DIS4 (p.Arg219Lys) has been reported to cause HypoPP2 by a mechanism distinct from gating pore current as the latter was too small to be predicted pathogenic (Kubota et al., 2020). Current density, activation and inactivation of Na_v1.4 were unaffected by p.Arg219Lys, but slow inactivation was not investigated. If these works demonstrate that Na_v1.4 LoF may result in hypokalemic episodes of muscle paralysis, it cannot be excluded that other genetics and/or environmental factors are important for their expressivity since they were not observed in familial forms.

The demonstration that Na_v1.4 LoF causes loss of muscle force has been provided in the last 5 years with the identification and the functional investigation of recessively-inherited SCN4A mutations in children with two forms of congenital muscle weakness: congenital myasthenic syndrome (CMS) and congenital myopathy (CM). Twenty-three recessively-inherited LoF mutations are now described in SCN4A (Figure 2 and Table 1). In contrast to the situation observed for dominantly-inherited Na_v1.4 channelopathies, no recurrent SCN4A mutations have been described for recessively-inherited Na_v1.4 channelopathies, the reported mutations being private. Both hypomorph (that causes a partial LoF) and null (that causes a complete LoF) mutations are observed.

Congenital myasthenic syndromes (CMS [MIM#608931]) form a clinically and genetically heterogeneous group of inherited disorders with skeletal muscle weakness that worsens with physical exertion (Engel et al., 2015; Nicole et al., 2017). They are caused by defective neurotransmission at the NMJ due to presynaptic (nerve terminal), synaptic cleft or most frequently post-synaptic (muscle) defects. Neurotransmission defects are documented by electromyographic testing showing a decrement of CMAP in response to repetitive nerve stimulation (RNS) at low (3 Hz) frequency or by single fiber electromyography (SFEMG) testing, a technique that detects neuromuscular transmission failure in motor units (Engel et al., 2015; Nicole et al., 2017). Thirty genes are known to cause CMS, which encode proteins that are critical for the formation, the maturation, the maintenance or the function of the NMJ such as post-synaptic nAChRs or proteins required for the synaptic aggregation of these receptors (agrin, Dok-7, rapsyn, MuSK, … ). CMS are most frequently recessively-inherited but dominant forms exist such as the slow-channel CMS that result from GoF mutations in the genes encoding nAChR subunits.

The first report of SCN4A mutations as the cause of fatigable muscle weakness suggestive of CMS was published nearly 20 years ago but the inheritance pattern (dominant or recessive) could not be established (Tsujino et al., 2003). This work reports one individual with abrupt paralytic attacks of respiratory and bulbar muscles in the neonatal period and general muscle weakness worsened by activity later in life. The patient was heterozygous for two SCN4A missense mutations (p.Ser246Leu and p.Val1442Glu). Electromyographic testing showed CMAP decrement at high (10 and 50 Hz) but not at low (2 Hz) RNS. One substitution (p.Val1442Glu) has strong LoF effects with enhanced fast inactivation, predicting that only 13% of Na_v1.4 channels are available for activation at the RMP. The second substitution (p.Ser246Leu) was concluded to be benign with mixed enhancing and impairing effects on slow inactivation. The inheritance pattern of the disease remained therefore unclear. The existence of recessively-inherited SCN4A mutations as the cause of fatigable muscle weakness has indeed been proven only recently. Four original articles have reported eight homozygous or compound heterozygous missense mutations in six unrelated individuals (Arnold et al., 2015; Habbout et al., 2016; Elia et al., 2019; Echaniz-Laguna et al., 2020). Symptoms suggestive of CMS with a decrement of CMAP at high (10–50 Hz; 2 out 6 patients tested) but not at low (3 Hz) RNS and jitter at SFEMG (2 out 2) were observed. They were combined to symptoms of PP, CM or even myotonia for p.Arg1460 substitutions. The decrement of CMAP in response to the long-exercise test was reported for 4 out 5 patients, and mild fiber size variabilities on muscle biopsies were observed for 2 out 4 examined.

Six of the eight SCN4A substitutions resulting in CMS are in or close to DIVS4, suggesting that this segment may be a hot-spot for SCN4A mutations causing fatigable muscle weakness (Figure 2). Four neutralize positively-charged residues and exert mixed GoF and LoF effects on fast inactivation (Table 1). They do not induce gating pore current in accordance with previous studies showing that the substitution of a single positively-charged residue in DIVS4 does not induce this current (Gosselin-Badaroudine et al., 2012). All the mutations investigated in patch-clamp experiments are LoF with reduced Na⁺ current density and/or enhanced fast or slow inactivation (Figure 4). Repetitive pulses at physiological frequencies (10–80 Hz) on heterologous cells induce a decrease of Na⁺ current amplitude for p.Val1442Glu, p.Arg1454Trp and p.Arg1457His mutant channels but not for WT or p.Ser246Leu channels (Figure 4) (Tsujino et al., 2003; Arnold et al., 2015; Habbout et al., 2016). Decrease of Na⁺ current in response to pulse trains (60–100 Hz) was on the contrary less pronounced for p.Arg1460Trp/Gln mutant channels than for WT channels (Elia et al., 2019). This may be due to the GoF effects of these variations, and may account for myotonia in individuals heterozygous for p.Arg1460Trp/Gln.

Congenital myopathies (CM) are another group of genetically and clinically heterogeneous diseases with muscle weakness (Gonorazky et al., 2018). There are many different types of CM with more than 30 known causative genes required for skeletal muscle development, structure or function. Most share common features, including lack of muscle tone and weakness. The degree of severity is wide, ranging from severe muscle weakness with in utero or neonatal lethality, to infant- or childhood-onset weakness (Wallgren-Pettersson and Laing, 2010). Several tests are performed to diagnose CM, including blood tests, electromyography, muscle biopsy and genetic testing. Fifteen recessive SCN4A mutations have been reported in nine unrelated kindreds with various severities of muscle weakness ranging from fetal hypokinesia (decreased or absent movements in utero) lethal at birth to “classical” CM for 11 individuals (Zaharieva et al., 2016; Gonorazky et al., 2017; Mercier et al., 2017; Sloth et al., 2018). Electromyographic investigations showed myopathic changes for some but not all individuals with classical CM (5 out 7). Marked fatigability with CMAP decrement at high (10Hz; 1 out 2 patients tested) but not at low (3 Hz) RNS was reported for some patients with “classical” CM (Zaharieva et al., 2016). When done, muscle biopsies showed fiber size variability, some necrotic myofibers and increased nuclear internalization but no specific structural abnormalities.

Mutations causing CM are observed on the entire length of Na_v1.4 (Figure 2; Table 1). Four are supposed to be null as they delete several segments. Four missense substitutions (p.Arg104His, p.Cys375Arg, p.Pro382Thr, p.Cys1209Phe) are functionally null as they do not produce any Na⁺ current in heterologous cells. Two are in one (DIS5S6) binding site for β1 subunit. Whether these missense mutations are null because of defective membrane trafficking or abnormal gating of the mutant channel is unknown. Four substitutions are hypomorph since mutant channels are still functional but have greatly reduced Na⁺ current density or impaired activation. One (p.Arg1135Cys) is reported to cause a recessively-inherited HypoPP2 phenotype and a gating pore current in vitro (Groome et al., 2014). Another mutation (p.Arg225Trp) has been reported to be heterozygous in one Korean patient with SCM but does not display GoF characteristics in vitro (Lee et al., 2009). One frameshift mutation (p.His1782Glnfs65) does not induce any gating defect in heterologous cells. This frameshift mutation results in the loss of the syntrophin binding site in the C-terminal tail of Na_v1.4 and its effect on the membrane expression of Na_v1.4 in myofibers remains to be determined. That strengthens the important notion that Na_v1.4 gating defects in vitro do not always fit the phenotypic expression. Moreover, the in vitro effects of pulse trains on Na⁺ current amplitude has not been tested for the SCN4A mutations causing CM.

Sodium Channel Weakness (SCW). LoF mutations that favor non-conducting (resting or inactivated) states of Na_v1.4 result in CMS, CM, and unusual forms of PP with hypokalemic episodes that are not due to a gating pore current (Figure 4). If PP, CMS and CM are distinct muscle disorders, the overlapping symptoms observed for individuals with Na_v1.4 LoF mutations suggest that they form a clinical spectrum with a continuum of membrane hypoexcitability as proposed for Na_v1.4 channelopathies due to GoF mutations (Figure 5) (Cannon, 2018). We propose here to name this spectrum “Sodium Channel Weakness (SCW)” as the clinical mirror of Sodium Channel Myotonia (SCM). A dosage-effect could occur in SCW that would explain the variable severity of muscle weakness: the fewer Na_v1.4 would be functional, the more severe would be the muscle weakness. Additional individuals with Na_v1.4 LoF mutations must be reported to ascertain this hypothesis important for genetic counseling and therapeutic issues. No specific biomarker (clinical signs, electromyographic testing or muscle biopsies investigations) has been identified for SCW, and the diagnosis of congenital muscle weakness due to Nav1.4 dysfunction requires to perform sequencing of SCN4A and to identify pathogenic variations.

One argument in favor of the dosage-effect hypothesis for SCW is the phenotype of Scn4a knock-out mouse mutants (Wu et al., 2016). Homozygous knock-out mice die at birth. Heterozygous mice have no gross locomotor deficits despite a half-reduced TTX-sensitive Na⁺ current recorded in dissociated myofibers. Loss of muscle force in response to factors inducing episodes of muscle weakness in PP was not observed in myofibers of heterozygous Scn4a ^+/- mice despite the reduced Na⁺ current (Wu et al., 2016). Scn4a haploinsufficiency has nevertheless a functional significance: if the tetanic force developed in response to supramaximal stimulus is like controls, a higher stimulating current density is required to achieve 50% maximal force in Scn4a ^+/- myofibers. Moreover, force decline is observed for Scn4a ^+/- myofibers in response to submaximal stimuli with higher sensitivity to partial blockade of neuromuscular transmission with curare (an antagonist of nAChR) compared to controls (Wu et al., 2016). This physiological work demonstrates that a half-reduction of functional Na_v1.4 channels in myofibers is not sufficient to induce PP by itself but reduces muscle force in challenging conditions. More recently, the lack of Na_v channels clustering at the NMJ in mice knocked-out for ankyrins has also been reported to cause muscle weakness with reduced spontaneous locomotor activity of homozygous mutant mice and CMAP decrements in response to 40 and 60 Hz RNS (Zhang et al., 2021). These two elegant studies confirm that the lack of synaptic Na_v1.4 or a half reduction in the whole amount of Na_v1.4 in myofibers results in muscle weakness with a fatigability component (Wu et al., 2016; Zhang et al., 2021).

Altogether, these studies suggest the following simplistic pathophysiological mechanism to account for muscle weakness when Na_v1.4 is deficient (Figure 6): LoF mutations would reduce the amount of Na_v1.4 channels available for activation by reducing their quantity or by altering their biophysical properties. Consequently, the genesis of muscle APs in response to epp and/or their propagation along the sarcolemma would fail more frequently than normal. This would result in a lower muscle AP frequency in the transverse tubules and so in a lower muscle force in response to motoneuronal firing. A fatigable component of the muscle weakness would result, at least in part, from enhanced inactivation properties that reduce Na_v1.4 availability for activation with repetitive stimulations.

To complete this overview of Na_v1.4 LoF in human diseases, a case-control genetic study reports more frequent SCN4A missense variants in infants deceased from sudden infant death syndrome (SIDS) than in controls (Mannikko et al., 2018). SIDS is a sudden, unexpected and unexplained death of an apparently healthy baby. The exact cause of SIDS is unknown and is thought to result from a combination of factors. Among the six SCN4A variants observed in a SIDS sample of 278 infants, four have a functional impact on Na_v1.4 current: two are GoF and two are LoF (p.Val1442Met in DIVS3S4 and p.Glu1520Lys in DIVS5S6). One (p.Val1442Met) substitutes one residue that induces SCW when substituted by Glu. An independent study performed in another sample of 73 infants with SIDS reported two inherited heterozygous missense variants (p.Lys724Arg in DIIS5S6 and p.Phe103Val in N-terminus) in SCN4A in two infants, but the biophysical characterization of the variant channels has yet to be done (Rochtus et al., 2020). The size of the two studied SIDS samples is low, and robust replicative studies are required to confirm Na_v1.4 dysfunction as a susceptibility factor for SIDS. In addition, several challenges are raised by these genetic association studies. The first is that the variant effects should lead to deleterious function in vivo. The second is the question of identifying infants with SCN4A variants and at risk for SIDS with all the bioethical issues raised by genetic testing.

These genetic association studies and the identification of Na_v1.4 LoF mutations as the cause of a large clinical spectrum of congenital muscle weakness further underline the influence of modifying factors on the phenotypic expression of SCN4A mutations already reported for GoF mutations (Nicole and Fontaine, 2015). For example, the p.Arg1451Leu substitution results in NDM (cold-induced myotonia), HyperPP, dominant PP with hypokalemic episodes or HypoPP2 with myotonia when homozygous, whereas p.Arg1135Cys results in recessive HypoPP2 or CM (Table 1) (Groome et al., 2014; Zaharieva et al., 2016; Luo et al., 2018; Poulin et al., 2018). The exact identity of the modifying factors is not known, although knowledge of muscle physiology makes ion channels (including trans- and cis- SCN4A variants), proteins important for the fine-tuning of Na_v1.4 channels and environmental factors strong candidates.

Several drugs are available as therapeutic tools against Na_v1.4 GoF for clinical use in humans (Jitpimolmard et al., 2020; Desaphy et al., 2021). Use-dependent Na_v blockers are efficient to reduce myofiber hyperexcitability and improve myotonic symptoms. The first line blocker is mexiletine with conclusive clinical trials and European orphan drug designation (Statland, 2012; Stunnenberg et al., 2018b). Therapeutic management of PP mostly relies on patient education with a life-style aimed to avoid triggering factors. Carbonic anhydrase inhibitors (acetazolamide and dichlorphenamide) also help to prevent paralytic episodes in PP, in addition to K⁺ supplement in HypoPP. Carbonic anhydrase inhibitors are known to prevent and improve attacks in HypoPP since several years but their mode of action is unclear. Acetazolamide is a diuretic drug used to treat several illnesses. It modulates multiple processes by inducing acidosis and by activating ion channels such as Ca²⁺-activated K⁺ (BK) and Cl⁻ channels, and probably acts by this means in PP (Eguchi et al., 2006; Tricarico et al., 2006). Bumetanide, an antagonist of NKCC, is also efficient to prevent hypokalemic-induced weakness in HypoPP1 and 2 mice (Wu et al., 2013a; Wu et al., 2013b). It acts by limiting the intracellular Cl⁻ concentration rise during repetitive APs, which helps to maintain the HypoPP fibers in the normal RMP when extracellular K⁺ concentration is low. Unfortunately, bumetanide was not efficient in a randomized controlled trial (RCT) pilot study performed on 10 individuals with HypoPP, a negative result that might be due to the small size of the sample. Molecules are also available to improve muscle force in CMS by modulating neuromuscular transmission (Engel, 2018). They include K⁺ channel blockers (3,4-diaminopyridine), acetylcholinesterase inhibitors (pyridostigmine), agonists of β2 adrenergic receptors (albuterol, ephedrine) for most forms of CMS and blockers of nAChRs for slow-channel CMS (quinidine, quinine, fluoxetine). No generic or specific treatments are available for CM, although several specific, especially gene-based, therapies are either in preclinical development or in early trials (Wallgren-Pettersson and Laing, 2010; Gonorazky et al., 2018).

Some but unfortunately not all individuals with SCW due to hypomorph SCN4A mutations benefited from pyridostigmine with acetazolamide or from acetazolamide alone (Arnold et al., 2015; Habbout et al., 2016; Elia et al., 2019; Echaniz-Laguna et al., 2020). Contrary to severe Na_v1.4 GoF conditions that greatly benefit from Na_v blockers (mexiletine, carbamazepine), there is no treatment reported to prevent early death linked to SCN4A LoF mutations in SCW (Gay et al., 2008; Lion-Francois et al., 2010).

Several inherited Na_v channelopathies are caused by LoF mutations such as Brugada syndrome, a cardiac arrhythmia due in part to Na_v1.5 gating defects, and Dravet syndrome, a severe and lifelong form of pediatric epilepsy due to Na_v1.1 haploinsufficiency (Loussouarn et al., 2015; Mantegazza et al., 2021). In all, a dosage effect is suggested, driving the therapeutic effort on boosting the mutant Na_v channels to eventually improve the clinical consequences of their LoF. The hypomorph nature of several SCN4A LoF mutations and the dosage effect suspected to occur in SCW suggest that such an approach would also be efficient for Na_v1.4 channelopathies (Figure 5).

Detailed pharmacological characterizations of Na_v activators are sparse compared to inhibitors and the current challenge fostered by the existence of LoF Na⁺ channelopathies is to identify Na_v channels agonists selective enough to be clinically useful. A complementary strategy to target-based drug approach would be to perform phenotype screening in order to identify molecules able to improve muscle weakness when Na_v1.4 is deficient (Swinney and Lee, 2020). The last few years have seen the impressive development of gene-based therapy for monogenic diseases with efficient vectors for selective gene delivery in vivo and the use of CRISPR-Cas9 technology for genome editing. Promising preclinical results have been obtained for some Na_v channelopathies, which could eventually benefit Na_v1.4 channelopathies.