Abstract

Rationale Hydrogen gas (H 2 ) produces pleiotropic therapeutic actions, but the exact molecular targets and ion-channel-based signaling cascades that underlie these benefits remain elusive. H 2 may regulate calcium ion (Ca 2+ )-dependent processes, but the direct involvement of H 2 in Ca 2+ signaling and its underlying molecular mechanisms are unknown. We propose that H 2 functions as a gaseous messenger that selectively opens a plasma-membrane Ca 2+ channel to evoke Ca 2+ transients ([Ca 2+ i ] t ) while avoiding cytotoxic overload, thereby offering a mechanism for its diverse biological effects. Methods This study employed real-time calcium imaging and CRISPR-Cas9 gene editing, with live-cell imaging to monitor real-time calcium signal intensity in living cells. Two-photon in vivo imaging was applied to detect real-time Ca 2+ signals in the brain and dorsal skin of C57BL/6 mice carrying adeno-associated virus-delivered calcium sensors. Live-cell F-actin staining and a wound healing (scratch) assay were used to assess the effects of H 2 on cell motility. Protein-protein docking and molecular dynamics simulations were performed to analyze the interaction interface and binding forces between TRPC4 and TRPC4AP in three-dimensional space. Additionally, RNA sequencing was performed to validate downstream biological effects and transcriptional regulation triggered by H 2 . Results H 2 elicited rapid and reversible [Ca 2+ i ] t across multiple cell types in a Ca 2+ - and concentration-dependent manner, an effect that was absent in TRPC4⁻/⁻ or TRPC4AP⁻/⁻ cells. In vivo imaging in mice expressing a genetically encoded Ca²⁺ sensor showed that H 2 inhalation elevated Ca 2+ signals in the motor cortex (M1 region) and dorsal skin. Functionally, live-cell imaging and wound-healing assays confirmed that H 2 -induced Ca 2+ transients enhanced cell motility. Mechanistically, protein docking revealed a dual-arginine cluster within the CIRB domain of TRPC4; its i