Enhancement of EFL-inactivation gate interaction by truncation of Nav1.5 carboxy-terminal domain.

Abstract

The voltage-gated sodium channel 1.5 (NaV1.5) participates in the initiation of the cardiac action potential by undergoing rapid activation which, within milliseconds, is followed by inactivation. Human channelopathic mutations in NaV1.5 are linked to various cardiac maladies, including cardiac arrythmia, atrial fibrillation, and dilated cardiomyopathies. The carboxy-terminal domain (CTD) of NaV1.5 is a well-recognized hotspot for channelopathic mutations. This domain harbors an EF-hand-like domain (EFL) and an IQ motif, which tune channel function via binding of auxiliary proteins, including the fibroblast growth factor homologous factors and calmodulin. In addition, the isolated NaV1.5 EFL has been reported to bind the channel inactivation gate (IG) via a partially buried hydrophobic pocket. However, whether this binding occurs in the full-length human channel and how it tunes NaV1.5 function are not well understood. Here, we have used high-resolution multi-channel electrophysiology recordings of disease-associated truncations and engineered NaV1.5 mutants to probe this interaction. We show that truncation of the NaV1.5 CTD increases late Na current, with a maximal increase observed for a truncation that includes helix V of the EFL (NaV1.5E[1864]∗), while further truncation resulted in reduced late Na current. Deletion of helix V increases the exposure of the EFL hydrophobic pocket, suggesting the large late Na current observed for NaV1.5E[1864]∗ may stem from a tighter interaction between the IG and the mutant EFL. Consistent with this, co-expression of NaV1.5E[1864]∗ with the WT NaV1.5 CTD or IG reduced late Na current, while expression with IG mutants designed to disrupt binding with either the pore-domain (IFM/QQQ) or EFL (K1504E/K1505E) showed only a partial reduction. These findings suggest that as NaV1.5 cycles between its functional states, the IG shuttles between the EFL and the pore domain shedding new light onto how these regions tune channel function.