Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

Voltage-dependent gating of the voltage-gated proton channels (H(V)1) 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 (delta pH). 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 reveal the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by delta pH. 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 H(V)1 channels.

Automated summary

Through patch-clamp fluorometry combined with the incorporation of fluorescent non-canonical amino acid Anap, we have obtained data on the voltage-dependent opening mechanism of H(V)1 channels. Our results suggest that motion of the S4 helix relative to an aromatic residue in the S2 helix leads to fluorescence increases upon depolarization, and Anap is quenched by this residue. Additionally, we found that the absolute value of the pH can impact the movement of the voltage sensor after channel opening.