Voltage Sensing in Hv1 Proton Channels

Abstract

Activation of the intrinsic aqueous water-wire proton conductance (GAQ) in Hv1 channels is controlled by changes in membrane potential and the transmembrane pH gradient (ΔpH). The mechanism by which changes in ΔpH affect the apparent voltage dependence of GAQ activation is not well understood. Recent work from our laboratory demonstrates that H+ current activation is rate-limited by a voltage-independent but ΔpH-sensitive transition when pHO ≤ pHI. In contrast, the Cole-Moore activation delay in Hv1 is relatively insensitive to changes in ΔpH, indicating that early steps in voltage sensor (VS) activation are distinct from later channel-opening transitions. In order to measure initial Hv1 voltage sensor activation in greater detail, we mutated a conserved Arg residue in the fourth helical segment (S4) to His and measured H+ currents under whole-cell voltage clamp in transfected HEK cells. Consistent with a previous results in the Shaker K+ channel mutant R362H (Starace and Bezanilla, 2004), we find that Hv1 R205H mediates a robust resting-state H+ ‘shuttle' conductance (GSH) at negative membrane potentials. The midpoint of the GSH-V relation is >100 more negative than GAQ-V and GSH gating kinetics are significantly faster than GAQ. Intriguingly, changes in ΔpH that are sufficient to produce a ∼+40 mV GAQ-V shift cause GSH-V to shift only ∼+10 mV. GSH gating thus reports a conformational change in Hv1 that occurs prior to the opening of GAQ and is differentially sensitive to changes in ΔpH. The data are consistent with the hypothesis that initial gating charge movement occurs early in the Hv1 activation pathway and is thermodynamically separable from aqueous H+ channel opening.