Voltage-Dependent Conformational Changes of the Voltage Sensor of KVAP Measured with LRET

Abstract

H v 1 voltage-gated proton channels appear to conduct H þ through a voltage sensor domain (VSD) that is homologous to that found in voltage-dependent cation channels and phosphatases. A conserved S4 transmembrane helix that contains a series of at least three Arg residues is integral to the voltage sensing function of all VSD proteins. In contrast to other VSD-containing proteins, voltagegated proton channels possess an additional unique biophysical property: coupling of the transmembrane pH gradient to voltage dependent activation. For both native voltage-gated H þ currents and expressed H v 1 channels, the apparent voltage threshold for H þ current activation (V thr ) shifts linearly ~40 mV per log([H þ ]) over at least five pH units. The molecular mechanism of coupling between voltage and the pH gradients represents one of the central mysteries of proton channel function. What constitutes the pH sensor in proton channels and how does it interact with the voltage sensor? DeCoursey and colleagues previously proposed a model for H þ channel gating wherein protonation of discrete sites that are alternatively exposed to either the extra-or intra-cellular milieu regulates the voltage-dependence of channel opening (Cherny et al., 1996); the required first step in this model is deprotonation of an extracellular H þ binding site. In order to identify residues that mediate pH-dependent regulation of voltage sensitivity in H v 1, we performed sitedirected mutagenesis to convert each of the candidate H þ acceptors in the H v 1 VSD to either neutral (alanine or asparagine), basic (arginine) or H þ -titratable (histidine) amino acids. Mutant channels were expressed in HEK-293 cells and V thr was determined under a variety of imposed pH gradients using whole-cell voltage clamp. Surprisingly, charge-neutralizing mutations failed to abrogate pH gradient sensing in H v 1. Our findings are interpreted in the context of the Cherny and DeCoursey model for proton channel gating.