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Interrogating the structure and function of the human voltage-gated proton channel (hHv1) with a fluorescent noncanonical amino acid

Year: 2026
Type: preprint
Abstract: The human voltage-gated proton channel (hH v 1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hH v 1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands, such as Zn 2+ . Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hH v 1, we developed a Förster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hH v 1 purified from E. coli . We used genetic code expansion (GCE) to generate a library of 14 full-length hH v 1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hH v 1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hH v 1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn 2+ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.
Source: eLife
DOI: 10.7554/elife.110161
Language: English
Open Access status: gold
Estimated APC paid: $2,500
Funders: Pew Charitable Trusts, University of Pennsylvania, Oregon State University, National Institutes of Health, National Eye Institute, National Institute of General Medical Sciences, National Institute of Diabetes and Digestive and Kidney Diseases
Awards: Gating Mechanisms of Retinal Cyclic Nucleotide-Regulated Ion Channels, Structural energetics of voltage- and ligand-dependent gating in ion channels, Conformational Energetics and Heterogeneity to Reveal Gating Mechanisms of TRPV and TRPM Ion Channels, Diabetes Research Center, Chemical Biology Probes to Decipher Regulation of TRPV1 in Living Cells, Vision Research Core
OpenAlex ID: W7131111897
Cites: 49
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Related to: 10