Molecular Basis of Voltage-Gated Proton Channel's Intracellular Trafficking

Abstract

Voltage-gated proton channel (HV1) presents a physiological role of great importance to phagocytosis and sub sequential death of microorganisms in phagocytic cells, such as neutrophils and microglia. Recently various studies describe the contribution of this channel in a higher malignity of tumors and its proliferation. Additionally, HV1 channels have been related to augmented tissue damage in cerebral stroke. Even though these pathological processes have been associated with a higher superficial expression of HV1 channels, little is known about how their abundance in the plasma membrane is regulated. Thus, by combining molecular approaches, in this work we have studied the molecular mechanisms of HV1 channel's intracellular trafficking, associating modifications in its C-terminal domain with its intracellular localization. Therefore, full length human voltage-gated proton channel (hHV1 WT) was fused with the fluorescent protein mCherry and site-directed mutagenesis were made in order to delete the C-terminal domain completely (V220X) or partially (I242X and I256X). Electrophysiological recordings of CHO-K1 cells transfected with hHV1 WT and the C-terminal truncated mutants showed that only the V220X mutant significantly reduced the proton current density. Thus, suggesting that the surface expression of this mutant is compromised. High-resolution microscopy analysis of hHV1 WT intracellular localization through co-transfection with 8 different intracellular organelles markers fused with EGFP resulted in a marked co-localization with Rab11, which is present in recycling endossomes. Furthermore, acute treatment of hHV1 WT transfected CHO-K1 cells with microtubule and actin disruptive agents, nocodazole and cytochalasin D respectively, diminished the proton current density significantly. Overall these results indicate an association of hHV1 WT with recycling endosomes, a key role of both cytoskeletal components and of the C-terminal domain in hHV1 forward trafficking.