Probing the Voltage Gated Proton Channel Hv1 with FRET

Abstract

The Hv1 proton channel has been shown to contribute to acid extrusion after acid loading in cells, it is involved in pH regulation in the airway epithelium and in the innate immune system during the respiratory burst and it has been proposed to play a role in the capacitation process of the sperm, aditionally the proton channel can also help set the membrane potential. The Hv1 channel is similar to the voltage sensing domain of voltage gated K+ and Na+ channels, it assembles into a dimer were each of the subunits forms a permeation pathway which is highly cooperative. Here, by measuring Foster Resonance Energy Transfer (FRET) between fluorescent proteins and with the non-fluorescent molecule dipicrylamine (DPA), we attempt to get some insigths about the organization and function of the Hv1 proton channel.mCitrine or mCerulean fluorescent proteins were attached to the C-termini of the channel. HEK 293 cells were cotransfected with the constructs and FRET was measured by the spectral FRET method. DPA was used as an energy acceptor for the mCitrine fluorescence in injected Xenopus oocytes membrane sheets. DPA intercalates in the membrane, quenching the fluorescence by a FRET mechanisms, allowing distance measurements between the membrane and the C-termini.We have been able to determine the relative separation between the C-termini of the dimer and its relation to the membrane plane. Results are consistent with the X-ray structure of the coiled-coiled C-terminal domain.This work is supported by grants from CONACYT No. 151297 and DGAPA-PAPIIT IN209209 to L.D.I.