Biochemical assessment of α-α-subunit interactions of Nav1.5 in a heterologous expression system

In cardiomyocytes, Na _v 1.5, the predominant voltage-gated sodium channel α-subunit, is localized at specialized membrane microdomains within the intercalated disc and lateral membrane. Although Na _v 1.5 remodeling within these microdomains could cause pathological cardiac phenotypes, it remains unclear whether Na _v 1.5 channels could form direct interactions or rather exist as individual proteins in close proximity within the cluster. Importantly, heterologous overexpression of any protein, and especially of the large transmembrane channel Na _v 1.5, could be associated with the insufficiency of endoplasmic reticulum folding machinery, hence leading to aspecific protein aggregation indistinguishable from the genuine α-α-subunit interactions. In this study, we show that the interactions between heterologous Na _v 1.5 proteins depend on nascent N-linked glycosylation, are supported by non-native intermolecular disulfide bonds, and are likely predisposed to hydrophobic “stickiness”. In addition, we show strong interactions between the full-length Na _v 1.5 and its truncated peptides: N-terminal domain, all four transmembrane domains, as well as the intracellular linker between domains I and II. Taken together, we conclude that the heterologous expression system is not optimal for the identification of α-α-subunit interaction sites of Na _v 1.5, and this study needs further investigation in native tissues.