Abstract
Voltage-dependent gating of the voltage-gated proton channels (HV1) remains poorly understood, partly because of the difficulty of obtaining direct measurements of voltage sensor movement in the form of gating currents. To circumvent this problem, we have implemented patch-clamp fluorometry in combination with the incorporation of the fluorescent non-canonical amino acid Anap to monitor channel opening and movement of the S4 segment. Simultaneous recording of currents and fluorescence signals allows for direct correlation of these parameters and investigation of their dependence on voltage and the pH gradient (ΔpH). We present data that indicate that Anap incorporated in the S4 helix is quenched by an aromatic residue located in the S2 helix, and that motion of the S4 relative to this quencher is responsible for fluorescence increases upon depolarization. The kinetics of the fluorescence signal reveals the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by ΔpH. Our experiments also suggest that the voltage sensor can move after channel opening and that the absolute value of the pH can influence the channel opening step. These results shed light on the complexities of voltage-dependent opening of human HV1 channels.
Significance statement The activation mechanisms of voltage-gated proton channels (HV1) are not well understood. Here we have combined patch-clamp fluorometry and a fluorescent non-canonical amino acid to uncover transitions in the activation pathway of human HV1 that are modulated by voltage and the pH gradient.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
New experiments have been added as well as new figures and Introduction and Discussion sections updated for clarity.
