Dynamic clustering and Rab11-dependent regulation of voltage-gated proton channels at the plasma membrane

Aims Voltage-gated proton channels (HV1) play a key role in intracellular pH homeostasis in immune cells and have been implicated in the neurological damage after a stroke and the prognosis and malignancy of several tumors. Thus, the molecular mechanisms governing HV1 surface abundance are of great clinical significance but have not yet been established.Materials and methods Human HV1 tagged with mCherry or EGFP was expressed in CHO-K1 cells. Channel organization and mobility were assessed by TIRF microscopy with single particle tracking, association with intracellular compartments by dynamic colocalization with fluorescently tagged markers, and channel function by whole-cell patch-clamp recording.Key findings HV1 channels form submicron clusters at and near the plasma membrane, which are mostly mobile and move predominantly by confined diffusion. This movement is independent of the cytoskeleton, although depolymerization of either network reduced proton current density and microtubule depolymerization additionally reduced cluster size. HV1 showed strong dynamic colocalization with Rab11, a recycling endosome marker. Co-expression of a dominant negative Rab11 increased proton current density but did not alter the cluster density, suggesting a modulatory role for Rab11 in channel activity.Significance Our work reveals a novel regulatory mechanism for HV1 channels in which dynamic clustering and Rab11 association alter channel activity with implications in disease contexts where HV1 is dysregulated.