gp91phox regulates Hv₁ channel gating and membrane trafficking via direct interaction

Voltage-gated proton channel (Hv₁) and the NADPH oxidase (NOX2) functionally cooperate to support ROS generation in immune cells through electrochemical coupling. However, whether NOX2 components directly regulate Hv₁ channel behavior remains unclear. In the present study, we investigated the molecular and functional relationship between Hv₁ and the NOX2 catalytic subunit gp91phox in BV2 microglial cells and the HEK293T expression system. Proximity ligation assay and co-immunoprecipitation studies revealed a physical interaction between Hv₁ and gp91phox. Electrophysiological experiments showed that endogenous Hv₁ currents in BV2 cells exhibited slow activation and deactivation kinetics, which were markedly accelerated following gp91phox knockdown. Conversely, co-expression of gp91phox with Hv₁ in HEK293T cells restored a slow-gating phenotype resembling native BV2 currents without significantly altering the voltage dependence of activation, indicating selective modulation of channel gating kinetics by gp91phox. Notably, deletion of gp91phox resulted in a dramatic (>80%) reduction in Hv₁ current density and membrane protein expression. Trafficking assays further demonstrated that gp91phox deficiency selectively destabilized membrane-associated Hv₁ without acutely affecting total cellular protein levels, indicating impaired channel trafficking. Collectively, these findings identify gp91phox as a direct regulator of Hv₁ channel function and membrane expression. Beyond classical electrochemical coupling, our study reveals a previously unrecognized role for NOX2 components in controlling Hv₁ channel gating and membrane trafficking, thereby establishing a novel mechanism coordinating redox signaling and proton channel regulation in microglial cells.