Mutant voltage-gated Na⁺ channels can exert a dominant negative effect through coupled gating

Abstract

Mutations in voltage-gated sodium channels are linked to several channelopathies leading to a wide variety of diseases including cardiac arrhythmias, epilepsy and myotonia. We previously demonstrated that Na1.5 trafficking-deficient mutant channels could lead to a dominant-negative effect by impairing trafficking of the wild-type (WT) channel. We also reported that voltage-gated sodium channels associate as dimers with coupled gating properties. Here, we hypothesized that the dominant-negative effect of mutant sodium channels could also occur through coupled gating. This was tested using cell surface biotinylation and single-channel recordings to measure the gating probability and coupled gating of the dimers. Co-expression of Na1.5-L325R with WT channels led to a dominant-negative effect as reflected by a 75% reduction in current density. Surprisingly, cell surface biotinylation showed that Na1.5-L325R mutant is capable of trafficking, with 40% of Na1.5-L325R reaching the cell surface when expressed alone. Importantly, even though a dominant-negative effect on the sodium current is observed when WT and Na1.5-L325R are expressed together, the total NaV channel cell surface expression was not significantly altered compared to WT alone. Thus, the trafficking deficiency could not explain the 75% decrease in INa. Interestingly, single-channel recordings showed that Na1.5-L325R exerted a dominant-negative effect on the WT at the gating level. Both coupled gating and gating probability of the WT:L325R dimers were drastically impaired. We conclude that dominant-negative suppression exerted by Na1.5 mutants can also be caused by impairing the WT gating probability, a mechanism resulting from the dimerization and coupled gating of voltage-gated sodium channel α-subunits.

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

“…Such homophilic interactions could explain the fact that several mutations in the Nav1.5 α‐subunit that underlie inherited cardiopathologies, act in a strongly dominant‐negative capacity, in which the mutant α‐subunit binds to and interferes directly with the trafficking and/or gating behavior of the wild‐type channel. Significantly, this occurs even when the mutant and wild‐type Nav1.5 channels are co‐expressed in HEK293F cells . The data presented here indicate that clustering may be an inherent property of Nav1.5 channels.…”

“…Previous evidence suggests that heterologously expressed Nav1.5 α‐subunits can assemble as dimers . The epitope recognized by the antibody used in our STORM experiments is located at the cytoplasmic C‐terminus of the Nav1.5 α‐subunit.…”

“…26 Previous evidence suggests that heterologously expressed Nav1.5 α-subunits can assemble as dimers. [27][28][29][30][31] The epitope recognized by the antibody used in our STORM experiments is located at the cytoplasmic C-terminus of the Nav1.5 α-subunit. By comparison with other Nav channels whose near atomic-resolution structure has been resolved, it is likely that this epitope lie close to or underneath the central pore of the channel, albeit with some flexibility.…”

Mutations in the Scn8a DIIS4 voltage sensor reveal new distinctions among hypomorphic and null Nav1.6 sodium channels

“…With the recent discovery that VGSC proteins can dimerize, it is also possible that the hypomorphic Δ9 or ∇3 alleles might exert a dominant‐negative effect on WT Na v 1.6 by forming mutant‐WT heterodimers . For instance, mutant isoforms of the VGSC Na v 1.5 have been reported to exert a dominant‐negative effect on sodium current via heterodimerization .…”

“…With the recent discovery that VGSC proteins can dimerize, it is also possible that the hypomorphic Δ9 or ∇3 alleles might exert a dominant‐negative effect on WT Na v 1.6 by forming mutant‐WT heterodimers . For instance, mutant isoforms of the VGSC Na v 1.5 have been reported to exert a dominant‐negative effect on sodium current via heterodimerization . Therefore, Na v 1.6 mutant‐WT heterodimers may also exhibit biophysical properties that are distinct from a WT homodimer, thereby contributing to the motor deficits and impaired acoustic startle response we observed in the Scn8a Δ9/+ and Scn8a ∇3/+ mutants.…”