Mechanism of pH sensing in the human voltage-gated proton channel hHv1

The voltage-gated proton channel Hv1 is indispensable for preserving physiological pH in cells and is characterized by distinct pH dependence. However, the mechanism of how Hv1 is regulated by pH is still obstacle. Under the framework of simulated hyperpolarized and depolarized states, continuous constant-pH molecular dynamics simulations have been performed to explore the coupling between conformational activation and protonation equilibra of the human Hv1. As a result, we identify E196 as the external pH sensor that works synchronously with the putative internal pH sensor H168. Further D174 is found to play a central role during the activation. Based on the pKa splitting between H140 and H193, we propose a pH-dependent mechanism of inhibitive Zn2+ binding to the external vestibule. Finally, our results offer an explanation of the measured threshold voltage shifts in response to the neutralizing mutations, including D174 above and three putative gating charges R205, R208 and R211, shedding light on the relationship between structure and function of the human Hv1.