Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.

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