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.

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.

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.

μ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50 values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3-Cys15, Cys4-Cys20 and Cys10-Cys21, respectively). Other analogues were not active enough for IC50 measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10-Cys21 is the most important for the NaV1.4 potency.