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Review
. 2021 Oct 4:12:751095.
doi: 10.3389/fphar.2021.751095. eCollection 2021.

New Challenges Resulting From the Loss of Function of Nav1.4 in Neuromuscular Diseases

Sophie Nicole  1   2 , Philippe Lory  1   2
Affiliations
Review

New Challenges Resulting From the Loss of Function of Nav1.4 in Neuromuscular Diseases

Sophie Nicole et al. Front Pharmacol. .

Abstract

The voltage-gated sodium channel Nav1.4 is a major actor in the excitability of skeletal myofibers, driving the muscle force in response to nerve stimulation. Supporting further this key role, mutations in SCN4A, the gene encoding the pore-forming α subunit of Nav1.4, are responsible for a clinical spectrum of human diseases ranging from muscle stiffness (sodium channel myotonia, SCM) to muscle weakness. For years, only dominantly-inherited diseases resulting from Nav1.4 gain of function (GoF) were known, i.e., non-dystrophic myotonia (delayed muscle relaxation due to myofiber hyperexcitability), paramyotonia congenita and hyperkalemic or hypokalemic periodic paralyses (episodic flaccid muscle weakness due to transient myofiber hypoexcitability). These last 5 years, SCN4A mutations inducing Nav1.4 loss of function (LoF) were identified as the cause of dominantly and recessively-inherited disorders with muscle weakness: periodic paralyses with hypokalemic attacks, congenital myasthenic syndromes and congenital myopathies. We propose to name this clinical spectrum sodium channel weakness (SCW) as the mirror of SCM. Nav1.4 LoF as a cause of permanent muscle weakness was quite unexpected as the Na+ current density in the sarcolemma is large, securing the ability to generate and propagate muscle action potentials. The properties of SCN4A LoF mutations are well documented at the channel level in cellular electrophysiological studies However, much less is known about the functional consequences of Nav1.4 LoF in skeletal myofibers with no available pertinent cell or animal models. Regarding the therapeutic issues for Nav1.4 channelopathies, former efforts were aimed at developing subtype-selective Nav channel antagonists to block myofiber hyperexcitability. Non-selective, Nav channel blockers are clinically efficient in SCM and paramyotonia congenita, whereas patient education and carbonic anhydrase inhibitors are helpful to prevent attacks in periodic paralyses. Developing therapeutic tools able to counteract Nav1.4 LoF in skeletal muscles is then a new challenge in the field of Nav channelopathies. Here, we review the current knowledge regarding Nav1.4 LoF and discuss the possible therapeutic strategies to be developed in order to improve muscle force in SCW.

Keywords: congenital myasthenic syndrome (CMS); congenital myopathy (CM); loss of function; skeletal muscle; sodium channel; therapeutics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Timeline highlighting important events for Nav1.4 channelopathies. PP: periodic paralysis; TTX: tetrodotoxin; HyperPP, hyperkalemic PP; HypoPP, hypokalemic PP; HypoPP2, hypokalemic PP, type 2; CMS, congenital myasthenic syndrome; cryo-EM, cryo-electron microscopy; NDM, non-dystrophic myotonia; LoF, loss of function; CM, congenital myopathy.
FIGURE 2
FIGURE 2
Structure of the pore-forming α subunit of human Nav1.4 and localization of the SCN4A loss-of-function (LoF) mutations in monogenic human disorders. (A) Schematic membrane topology of Nav1.4 α subunit with four domains (DI-DIV), each domain being composed of six transmembrane segments (S1-S6). The voltage-sensor S4 segments are rich in positively-charged amino acid residues (+). The LoF mutations are shown. Hypokalemic periodic paralysis (HypoPP), orange stars; periodic paralysis (PP) with hypokalemic episodes, yellow stars; CMS, green (missense mutation) and red (nonsense or frameshift mutation) circles; CM, blue (missense mutation) and red (nonsense or frameshift mutation) squares. The sites of binding interactions with the transmembrane β1 subunit, and the cytoplasmic ankyrin, calmodulin and syntrophin proteins are indicated. (B) Alignment of amino acid sequences of the four S4 segments of human Nav1.4 with positively-charged residues (in bold). The gating charges are named R1 to R4. The position of the missense mutations causing HypoPP2 (orange), PP with hypokalemic episodes (yellow), CMS (green) and CM (blue) is indicated. Underlined residues cause different phenotypes when substituted: dominant NDM or recessive CM (p.Arg225); de novo HypoPP2, recessive HypoPP2 or CM (p.Arg1135); dominant NDM, glucocorticoid-induced HypoPP, and dominant or recessive PP with hypokalemic episodes (p.Arg1451). To note that the substitutions of p.Arg675 (R3, DIIS4) and p.Arg1135 (R3, DIIIS4) cause HypoPP2 or normokalemic PP with corticosteroid- or thyrotoxicosis-induced hypokalemic episodes of paralysis and depolarization- and not hyperpolarization-activated gating pore current (Vicart et al., 2004; Sokolov et al., 2008; Groome et al., 2014).
FIGURE 3
FIGURE 3
Nav channels, Na+ current and neuromuscular junctions. (A) Left: a phase-contrast image of an adult mouse Levator aureus longus (LAL, fast-twitch) muscle. The myelinated nerve (arrows) terminates at neuromuscular junctions (NMJs, stars) (scale bar, 15 μm). Right: examples of Na+ current recordings made with a loose-patch clamp electrode from the extrasynaptic membrane (extrajunctional, top trace) and synaptic (endplate, bottom trace) membrane of a LAL myofiber. The extrasynaptic Na+ current density is equal to 23.3 mA/cm2 and the endplate Na+ current density is equal to 110 mA/cm2 (adapted from (Lupa et al., 1993); Copyright [1993] Society for Neuroscience). (B) Confocal image of fluorescent staining of nAChRs (stained with α bungarotoxin, in red in the merged image), Nav channels (anti-pan antibody binding to all Nav isoforms, in green in the merged image), and nerve (anti-NF 200 and anti-synaptophysin antibodies, in blue in the merged image) on dilacerated Tibialis anterior (fast-twitch) myofibers from an adult mouse (X 63). (C) Schema of one NMJ with its presynaptic (nerve terminal rich in synaptic vesicles, up) and post-synaptic (myofibers with post-synaptic folds, down). AChRs (red) are clustered at the top and Nav channels (Nav1.4 in green) are clustered in the depth of the postsynaptic folds. The synaptic aggregation of Nav1.4 would result from its binding interaction with ankyrins (purple), themselves linked to spectrin (deep blue). Nav1.4 would interact with syntrophin (brown) in the extrasynaptic sarcolemma.
FIGURE 4
FIGURE 4
Gating of Nav1.4 with sodium channel weakness (SCW) mutations. (A) Illustrative Na+ current traces for wild-type (WT, black) and mutant (LoF, green) Nav1.4 channels obtained in HEK-293 cells. Differences include reduced current density, impaired activation, and enhanced inactivation for hypomorph mutants (dark green). No current is recorded for null mutants (light green). (B) Three-states model of Nav1.4 channels. The resting or inactivated states are favored in SCW. (C) Relative Na+ current amplitude (pulse 50 compared to pulse 1) is observed in response to pulse trains at 10, 20 and 50 Hz for the mutant (LoF, green) but not the WT (black) channels in heterologous cells (adapted from Habbout et al., 2016). (D) Main differences between dominantly-inherited HypoPP2 mutations and recessively-inherited SCW mutations substituting Arginine (Arg) residues in S4 segments of Nav1.4. The gating pore current induces a dominant-negative (DN) effect on central Nav currents in HypoPP2. Loss of function (LoF) occurs in both.
FIGURE 5
FIGURE 5
Clinical spectrum of Nav1.4 channelopathies with a continuum from membrane hyperexcitability in sodium channel myotonia (SCM) to hypoexcitability in sodium channel weakness (SCW), effect of the mutations on Nav1.4 gating and available as well as potential (?) therapeutic options.
FIGURE 6
FIGURE 6
Schematic representation of a simple pathophysiological hypothesis to account for sodium channel weakness (SCW) due to Nav1.4 loss of function (LoF). Repetitive muscle action potentials (myofiber AP) resulting from motoneuronal firing lead to the summation of muscle twitches (contractile activity), in this example up to the maximal sustained contraction (tetanos) in wild-type muscles (WT Nav1.4, black traces). Nav1.4 LoF would reduce the amount of Nav1.4 channels available for activation. This would result in lower muscle AP frequency and muscle force in response to neuronal firing in mutant myofibers (LoF Nav1.4, green). The fatigability would result from a decrease of Nav1.4 availability during neuronal firing, which would progressively reduce muscle AP frequency and muscle force.
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