Project Summary Voltage-gated ion channels are a diverse group of membrane proteins that play significant roles in a variety of physiological and pathological processes, from neuronal excitability and muscle contraction, to autoimmunity, stroke, and cancer. They all share a common structural module, the voltage-sensing domain (VSD), responsible for turning on and off an effector domain in response to changes in membrane potential. Previous studies from us and other groups have shown that, while most VSDs do not conduct ions, they can become leaky as a result of mutations. Mutated VSDs permeable to ions or protons are responsible for serious genetic disorders, such as hypokalemic periodic paralysis, and cardiac arrhythmias with dilated cardiomyopathy. The VSD of the voltage-gated channel Hv1, on the other hand, is inherently proton-conductive and this property is key to the channel's many physiological functions. The long-term goal of this study is to elucidate how VSDs conduct ions and protons, how their activity is regulated, and how they can be blocked pharmacologically for therapeutic purposes. Here, we will focus on the Hv1 channel, an emerging drug target for a variety of diseases, including cancer and stroke. The mechanism underlying VSD-mediated proton conduction in Hv1 is poorly understood and there is an unmet need for small-molecule inhibitors of Hv1 activity. We have previously discovered a class of compounds that act as Hv1 blockers and characterized their binding environment. We identified aromatic interactions within the core of the channel's VSD that could be harnessed to create better drugs to suppress Hv1 activity. In aim 1, we propose to use electrophysiological measurements and unnatural amino acid substitutions to examine how these interactions contribute to Hv1 block and voltage-dependent activation. One of the main problems limiting our understanding of proton-selective permeation is the inadequate description of channel- proton interactions by simulation methods based on classic mechanics. In aim 2, we will use quantum mechanics/molecular mechanics simulations on a validated Hv1 structural model in combination with the rational design of a proton-conducting VSD to obtain detailed information on how protons move within the Hv1 permeation pathway. Hv1 function is known to be tightly regulated in the cell. But, little is known about how this regulation is achieved. We have recently identified a new modality of channel regulation mediated by mechanical stress, which can provide an explanation for the hyperactivity of Hv1 previously described in microglia under conditions of ischemic stroke. In aim 3, we will use electrophysiology, high-speed pressure clamp stimulation, and targeted mutagenesis to determine the mechanism of Hv1 mechanosensitivity.