Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid
- Universidad Nacional Autónoma de México, Mexico
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 (DpH). 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 DpH. 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.
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All data generated or analysed during this study are included in the manuscript and supporting file.
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Funding
Programa de Doctorado en Ciencias Bioquimicas-UNAM (CONACyT No. 463819 (CVU 659182))
- Esteban Suárez-Delgado
Programa de Doctorado en Ciencias Bioquimicas-UNAM (CONACyT No. 788807 (CVU 1101710))
- Maru Orozco-Contreras
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Suárez-Delgado et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid
eLife 12:e85836.
https://doi.org/10.7554/eLife.85836
