Membrane Topology of S4 of the Mouse Voltage-Gated Proton Channel

Abstract

VSOP/Hv1 is a voltage-gated proton channel that contains the voltage sensor domain (VSD) but not pore domain. VSD of VSOP/Hv1 allows protons to permeate as well as sensing voltage. It has been reported that basic amino acids in the fourth transmembrane segment (S4) of voltage-gated ion channels play critical roles in voltage-sensing. Mouse VSOP (mVSOP) has three arginine residues (R1, R2, R3) in a pattern similar to those conserved in other voltage-gated channels. To address the role of S4 in mVSOP, we have reported that the truncated construct (A206sop) just downstream of R2 in the S4 is still ion-conductive (Biophysical Society 53th Annual Meeting, 2009). In this study, we further analysed properties of A206stop. The outward current of A206stop was almost completely blocked by zinc. Visualization of intracellular pH using BCECF (pH-sensitive ratiometric dye) showed that cytoplasm of tsA201 cell was alkalinized under the depolarization condition. Na and K ion do not permeate through A206stop. Gating properties of the proton currents through A206stop were sensitive to either intracellular pH or extracellular pH. However, voltage dependency of A206stop was weaker than that of full-length mVSOP, and the I-V relationship of A206stop was shifted rightward. These results indicate that A206stop retains the basic properties of the voltage-gated proton channel even if it lacks a half of S4. We also carried out two biochemical assays: site-directed cysteine-scanning using accessibility of maleimide-reagent as detected by western blotting (pegylation protection) and in vitro glycosylation assays. Both showed that S4 of A206stop inserts into the membrane and the position of A206 faces intracellular aqueous environments. These findings suggest that the region downstream of the R2 position of S4 of VSOP/Hv1 is not essential for proton selectivity.