The Resting State of Human Proton Channel from Functional and Structural Determinations

Abstract

The human voltage gated proton channel (Hv1) plays an important role in a wide variety of physiological functions, including male fertility, immune response and metastatic proliferation. Upon transmembrane voltage stimulus, the rearrangement of positively charged S4 leads to highly selective and directional conduction of proton. Hv1 carries out this function on the structural blueprint of the S4-containing voltage sensing domains (VSD). As a way to understand Hv1 gating, we have developed a simple biochemical preparation of functional Hv1 as shown by established fluorescence based proton flux assay. We have probed Hv1 in the functional proteoliposome by site-directed spin labeling EPR spectroscopic (SDSL-EPR) methods. Mobility and accessibility information show that the secondary structure of transmembrane segments is generally consistent with the expected VSD topology. Solvent accessibility measurements revealed that the gating Arginines (205, 208 and 211) are located on the lower part of S4, the internal leaflet of the bilayer. The presence of a narrow solvent occluded region strongly suggests a short proton conduction pathway, and does not seem to be compatible with the idea of a long water wire across membrane. Furthermore, the Hv1 represents a resting state VSD at 0 mV according to its G-V curve . The structural details from Hv1 provide unique insight to not only the proton conduction, but also the mechanism of voltage sensing.