PH Dependence of a Monomeric Non-Conducting Voltage-Gated Proton Channel (HV1)

Abstract

The voltage-gated proton channel (Hv1) is a transmembrane protein made of a dimer of four transmembrane segments. This structure is capable of sensing the voltage and conforms the permeation pathway to protons. One of the most striking biophysical properties of Hv1 is its pH dependence of gating. The conductance-voltage (G-V) curve of Hv1 shifts depending on the pH gradient established across the membrane, but the molecular mechanism of this property is still poorly understood. Recently, gating currents of Hv1 were characterized using the low-conducting N264R mutant, giving us a new tool to understand the mechanism behind the pH dependence of Hv1. We measured currents produced by the monomeric N264R mutant of Ciona intestinalis Hv1 at different internal and external pH in membrane patches of Xenopus laevis oocytes. Although results showed a clear pH dependence of the gating currents, the presence of proton currents and charge trapping produced by the N264R mutation prevent a detailed analysis of the data. To eliminate proton currents, we measured gating currents produced by a monomeric non-conducting Hv1. Gating currents of the non-conducting mutant are free of proton current contamination and charge trapping. They preserve all the characteristics of Hv1 gating currents described previously. The charge-voltage (Q-V) curve is shifted to the left at lower ΔpH (pHin-pHout). Additionally, the ON-gating current decay is faster when ΔpH decreases. Our results show that the non-conducting mutant is a better choice to study pH dependence of Hv1 and understand the molecular mechanism behind this biophysical property. Supported by CONICYT-PFCHA/Doctorado Nacional/2017-21170395 to E.C., Fondecyt 1180464 to C.G. and 1150273 to R.L. The Centro Interdisciplinario de Neurociencia de Valparaiso is a Millennium Institute supported by the Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism (P029-022-F).