Inhibition of Voltage-Gated Hv1 Channel by Guanidine Derivatives

Abstract

The voltage-gated proton channel Hv1 plays a key role in the regulation of ROS production by NOX enzymes in phagocytic cells. Excessive channel activity enhances proliferation and invasiveness in cancer cells, and worsens brain damage after ischemic stroke. The Hv1 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 the Hv1 channel and that its binding site in the VSD is accessible from the intracellular side of the membrane only when the channel is open. Here, we examine the apparent binding affinities of a series of 2GBI derivatives on Hv1 channels mutated at positions located in the core of the VSD and apply mutant cycle analysis to determine the most likely interactions between channel and inhibitor. We identify four residues involved in 2GBI binding and determine the orientation of the blocker inside the VSD. Our data provide a simple explanation for the very large difference in binding affinity between 2GBI and the related compound 2-guanidinobenzoxazole (2GBOZ), and suggest ways to design more effective Hv1 inhibitors. We also investigate the molecular characteristics that allow guanidine derivatives to reach the binding site when added from the extracellular side of the membrane so that they can be used to block endogenous proton channels under physiological conditions. This work is supported by NIH -National Institute of General Medical Sciences, grant R01GM098973.