Possible Interactions of Extracellular Loop IVP2-S6 With Voltage-Sensing Domain III in Cardiac Sodium Channel.

Anastasia K Zaytseva,Boris S Zhorov,Anna A Kostareva,Aleksandr S Boitsov

doi:10.3389/fphar.2021.742508

Abstract

Motion transmission from voltage sensors to inactivation gates is an important problem in the general physiology of ion channels. In a cryo-EM structure of channel hNav1.5, residues N1736 and R1739 in the extracellular loop IVP2-S6 approach glutamates E1225 and E1295, respectively, in the voltage-sensing domain III (VSD-III). ClinVar-reported variants E1230K, E1295K, and R1739W/Q and other variants in loops IVP2-S6, IIIS1-S2, and IIIS3-S4 are associated with cardiac arrhythmias, highlighting the interface between IVP2-S6 and VSD-III as a hot spot of disease mutations. Atomic mechanisms of the channel dysfunction caused by these mutations are unknown. Here, we generated mutants E1295R, R1739E, E1295R/R1739E, and N1736R, expressed them in HEK-293T cells, and explored biophysical properties. Mutation E1295R reduced steady-state fast inactivation and enhanced steady-state slow inactivation. In contrast, mutation R1739E slightly enhanced fast inactivation and attenuated slow inactivation. Characteristics of the double mutant E1295R/R1739E were rather similar to those of the wild-type channel. Mutation N1736R attenuated slow inactivation. Molecular modeling predicted salt bridging of R1739E with the outermost lysine in the activated voltage-sensing helix IIIS4. In contrast, the loss-of-function substitution E1295R repelled R1739, thus destabilizing the activated VSD-III in agreement with our data that E1295R caused a depolarizing shift of the G-V curve. In silico deactivation of VSD-III with constraint-maintained salt bridge E1295-R1739 resulted in the following changes: 1) contacts between IIIS4 and IVS5 were switched; 2) contacts of the linker-helix IIIS4-S5 with IVS5, IVS6, and fast inactivation tripeptide IFM were modified; 3) contacts of the IFM tripeptide with helices IVS5 and IVS6 were altered; 4) mobile loop IVP2-S6 shifted helix IVP2 that contributes to the slow inactivation gate and helix IVS6 that contributes to the fast inactivation gate. The likelihood of salt bridge E1295-R1739 in deactivated VSD-III is supported by Poisson–Boltzmann calculations and state-dependent energetics of loop IVP2-S6. Taken together, our results suggest that loop IVP2-S6 is involved in motion transmission from VSD-III to the inactivation gates.

Highlights

Voltage-gated sodium channels play key roles in the physiology and pathophysiology of excitable cells (Ahern et al, 2016; Catterall, 2017; Schwartz et al, 2020)
Our results suggest that loop IVP2S6 is involved in motion transmission from voltage-sensing domain (VSD)-III to the inactivation gates
The selectivity filter divides the ion permeation pathway into two parts: the outer pore exposed to the extracellular space and the inner pore, which merges with the cytoplasm in the open pore domain (PD)

Summary

INTRODUCTION

Voltage-gated sodium channels play key roles in the physiology and pathophysiology of excitable cells (Ahern et al, 2016; Catterall, 2017; Schwartz et al, 2020). The activation gate, which is formed by hydrophobic residues at the C-terminal halves S6s, opens and sodium ions flow into the cell. A few milliseconds after activation, the sodium channel transits to the fast inactivation state due to binding of the IFM tripeptide in the III/IV linker to the hydrophobic cleft between helices IIIS5, IIIS6, IVS5, and IVS6. In all available cryo-EM structures of the Nav1.x channels, a semirigid 15-membered disulfide-fastened loop IVP2-S6 hangs over the voltage-sensing domain III (VSD-III). Many ClinVarreported disease mutations of Nav1.5 are located at the interface between VSD-III and extracellular loop IVP2-S6, implying functional importance of the interface. In the cryo-EM structure of the hNav1.5 channel (PDB ID: 6lqa), residues E1230 and E1295 in the VSD-III approach, respectively, N1736 and R1739 in loop IVP2-S6. Our experimental data and molecular models suggest that electrostatic interactions between VSD-III and loop IVP2-S6 may contribute to motion transmission from VSD-III to the SF gate at the N-end of helix IVP2 and the fast inactivation gate at the C-end of helix IVS6

METHODS

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT