Project Summary/Abstract Voltage-gated proton (Hv) channels carry robust proton currents across membranes and are gated by both voltage and transmembrane pH gradient (∆pH). They normally serve as proton extruders to maintain the pH homeostasis of metabolically active cells. In phagocytes, human Hv1 (hHv1) channels compensate for charge and pH imbalance during the respiratory burst of NADPH oxidase to promote the production of reactive oxygen species (ROS) for pathogen defense. The sperm hHv1 channels trigger intracellular alkylation essential for capacitation. The hHv1 channel also highly correlates with cancer invasiveness and ischemic neuronal cell death. Voltage and ∆pH gating are two fundamental biophysical properties determining the dynamics of proton currents through Hv channels, which in turn underlie their physiological and pathophysiological roles in the cells mentioned above. Funded by the parent award, we examined the conformational dynamics of the purified hHv1 proteins in liposomes using single molecule FRET (Fluorescence Resonance Energy Transfer). We have provided the first glimpse of real-time conformational transitions in the hHv1 voltage sensor and showed that both voltage and pH gate the hHv1 channel by modifying the conformational landscapes of the voltage sensor. We also generated a kinetic model to explain how voltage pH interplay determines hHv1 channel gating. To maximize the impacts of the exciting findings that have been made, we need to obtain accurate rate constants of conformational transitions described by the kinetic model, which can provide key mechanistic insights into the voltage sensing and gating in other voltage-gated cation channels. The administrative supplement for the accessory will upgrade the existing TIRF (Total Internal Reflection Fluorescence) microscope for single molecule FRET imaging, which will provide the critical technical strength to maximize the scientific impacts of the parent award. The accessory contains the patch-clamp module for electrophysiological recording and the single molecule FRET imaging module containing a high-speed sCMOS (scientific Complementary Metal- Oxide-Semiconductor) camera reaching a time resolution close to 1 millisecond. With the accessory, the existing TIRF microscope will be upgraded to perform single molecule FRET imaging and electrophysiological recordings simultaneously. As a result, we will be able to control the voltage and pH applied to hHv1 channels more precisely to get accurate rate constants of the conformational transitions in the hHv1 channel induced by pH and voltage. In addition, we will be able to examine the channel gating dynamics by patch-clamp recordings and conformational dynamics using single molecule FRET imaging simultaneously, thus defining the structure and function relationship directly.