TY - JOUR TI - Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid AU - Suárez-Delgado, Esteban AU - Orozco-Contreras, Maru AU - Rangel-Yescas, Gisela E AU - Islas, Leon D A2 - Pless, Stephan A A2 - Aldrich, Richard W VL - 12 PY - 2023 DA - 2023/01/25 SP - e85836 C1 - eLife 2023;12:e85836 DO - 10.7554/eLife.85836 UR - https://doi.org/10.7554/eLife.85836 AB - 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 reveal 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. KW - proton channels KW - fluorescence KW - non-canonical amino acids KW - channel gating KW - HEK293 cells JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -