Exploring Noncanonical Coupling in Voltage-Gated Sodium Channels

Abstract

Eukaryotic voltage-gated sodium channels (Nav) contain four homologous domains I-IV (DI-IV), each is comprised of six transmembrane segments (S1-S6) analogous to a voltage-gated potassium channel (Kv) subunit. A common structural architecture shared between the Nav and many Kv channels is the domain-swapped assembly, which results in interfaces between voltage sensor domains (VSD) and pore domains (PD) of different domains. The VSD enables channel sensitivity to voltage. The canonical mechanism that couples the VSD to the PD includes the S4-S5 linker that ultimately links movements of the segment S4 VSD to the segment S6 helix of the PD. A noncanonical pathway comprised of a noncovalent network connects the VSD to the PD in the Shaker K+ channel. Here, we aimed to elucidate the possibility of a noncanonical pathway in Nav1.4. Single point mutations were introduced into positions of Nav1.4 that have structural analogs to the VSD-PD pathway that are known to disrupt the noncanonical pathway in Shaker. We studied L1445H (DIV-S4), V267W (DI-S5), and L266A (DI-S5), which are analogous to positions L366, S411 and S412, respectively, in Shaker. Initially, Nav1.4 was expressed without the β-subunit in Xenopuslaevis oocytes. Compared to the wild-type, the mutants had the same voltage dependence in conductance (G-V) and showed a slight voltage shift in steady-state fast inactivation. The time constants of fast inactivation settling were two to three times slower. To study steady-state slow inactivation, a 60 s depolarizing pulse was followed by a recovery interval for fast inactivation (20 ms hyperpolarizing pulse) and tested with a 0 mV pulse. The mutants showed a clear enhancement of the steady-state slow inactivation. Together, the data indicates that perturbations introduced into the Nav1.4 DIV-S4 and DI-S5 interface have an impact on the channel's function. (Support: NIHGM030376)