Abstract

The human Hv1 (hHv1) channel adopts a dimeric assembly, and each protomer shares homology with the voltage-sensing domain of voltage-gated K + and Na + channels. By responding to membrane depolarization and cross-membrane pH gradient, Hv1 channels play important physiological roles especially in acid extrusion. Here we report that hHv1 is highly sensitive to singlet oxygen ( 1 O 2 )-medicated photodynamic modification (PDM). In the presence of a positive pH gradient, PDM accelerates hHv1 activation and slows deactivation, and minimally shifts in the conductance-voltage curve. The involvement of 1 O 2 is supported by the use of known photosensitizers, including the singlet oxygen photosensitizing protein-3 (SOPP3) and rose Bengal, and two 1 O 2 quenchers. Alanine replacement of a histidine residue, H168, which is important for sensing intracellular pH, completely abolishes the PDM effects. PDM disrupts cooperative gating between two protomers by transforming the channel kinetics into a single exponential; conversely, monomeric hHv1, made by removing the C-terminal cytoplasmic region, does not appear to respond to PDM. Finally, in the absence of a positive pH gradient, PDM exerts a more dramatic impact on WT hHv1-SOPP3 gating, in contrast to the non-significant effects on H168A and ΔC-deletion mutant channels. This study establishes PDM of hHv1 and provides mechanistic insights into the concerted actions of voltage, pH gradient, dimeric assembly, and 1 O 2 -mediated modification on channel gating.