Abstract
The voltage-gated proton channel Hv1 plays important roles in proton extrusion, pH homeostasis, and production of reactive oxygen species in a variety of cell types. Excessive Hv1 activity increases proliferation and invasiveness in cancer cells and worsens brain damage in ischemic stroke. The channel is composed of two subunits, each containing a proton-permeable voltage-sensing domain (VSD) and lacking the pore domain typical of other voltage-gated ion channels. We have previously shown that the compound 2-guanidinobenzimidazole (2GBI) inhibits Hv1 proton conduction by binding to the VSD from its intracellular side. Here, we examine the binding affinities of a series of 2GBI derivatives on human Hv1 channels mutated at positions located in the core of the VSD and apply mutant cycle analysis to determine how the inhibitor interacts with the channel. We identify four Hv1 residues involved in the binding: aspartate 112, phenylalanine 150, serine 181, and arginine 211. 2GBI appears to be oriented in the binding site with its benzo ring pointing to F150, its imidazole ring inserted between residue D112 and residues S181 and R211, and the guanidine group positioned in the proximity of R211. We also identify a modified version of 2GBI that is able to reach the binding site on Hv1 from the extracellular side of the membrane. Understanding how compounds like 2GBI interact with the Hv1 channel is an important step to the development of pharmacological treatments for diseases caused by Hv1 hyperactivity.