Abstract
Voltage-gated proton channels (Hv1) are ubiquitous throughout nature and are implicated in numerous physiological processes. The gene encoding for Hv1, however, was only identified in 2006. The lack of sufficient structural information of this channel has hampered the understanding of the molecular mechanism of channel activation and proton permeation. This study uses both simulation and experimental approaches to further develop existing models of the Hv1 channel. Our study provides insights into features of channel gating and proton permeation pathway. We compare open- and closed-state structures developed previously with a recent crystal structure that traps the channel in a presumably closed state. Insights into gating pathways were provided using a combination of all-atom molecular dynamics simulations with a swarm of trajectories with the string method for extensive transition path sampling and evolution. A detailed residue–residue interaction profile and a hydration profile were studied to map the gating pathway in this channel. In particular, it allows us to identify potential intermediate states and compare them to the experimentally observed crystal structure of Takeshita et al. (Takeshita K, Sakata S, Yamashita E, Fujiwara Y, Kawanabe A, Kurokawa T, et al. X-ray crystal structure of voltage-gated proton channel. Nature 2014). The mechanisms governing ion transport in the wild-type and mutant Hv1 channels were studied by a combination of electrophysiological recordings and free energy simulations. With these results, we were able to further refine ideas about the location and function of the selectivity filter. The refined structural models will be essential for future investigations of this channel and the development of new drugs targeting cellular proton transport.
Our study reports on basic biophysical principles governing selective ion permeation in voltage-gated proton channels, which are membrane proteins with important roles in immune response and fertility. To further confirm and develop our model, we compared it to recently reported crystal structures. To gain further insight into the mechanisms of ion selectivity in these channels, we performed in vitro and in silico mutations on the channels and investigated their functioning. We found that targeted modifications around the constriction zone formed in an open state of the channel dramatically affect ion selectivity of the channel enabling transport of Na+. We also further investigate the gating behavior of the wild-type structures. Our in silico predictions were confirmed experimentally with regard to both the mutant and the wild-type structures, further establishing the validity of the channel model for future applications in drug development targeting this voltage-gated proton channel. [Display omitted]
