Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx

Hydrogen gas (H₂) produces pleiotropic therapeutic actions, but the exact molecular targets and ion-channel-based signaling cascades that underlie these benefits remain elusive. H₂ may regulate calcium ion (Ca²⁺)-dependent processes, but the direct involvement of H₂ in Ca²⁺ signaling and its underlying molecular mechanisms are unknown. We propose that H₂ functions as a gaseous messenger that selectively opens a plasma-membrane Ca²⁺ channel to evoke Ca²⁺ transients ([Ca²⁺ _i]_t) while avoiding cytotoxic overload, thereby offering a mechanism for its diverse biological effects.

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 Ca²⁺ 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 H₂ 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 H₂.

H₂ elicited rapid and reversible [Ca²⁺ _i]_t across multiple cell types in a Ca²⁺- 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 H₂ inhalation elevated Ca²⁺ signals in the motor cortex (M1 region) and dorsal skin. Functionally, live-cell imaging and wound-healing assays confirmed that H₂-induced Ca²⁺ transients enhanced cell motility. Mechanistically, protein docking revealed a dual-arginine cluster within the CIRB domain of TRPC4; its interaction with TRPC4AP was essential for H₂-evoked Ca²⁺ influx. Mutating these arginines to alanine residues completely abolishing the response. H₂ triggered proton efflux and increased intracellular pH. Molecular dynamics simulations indicated that altered pH modulates the binding force between TRPC4 Arg730/Arg731 and TRPC4AP. Transcriptomic analysis further demonstrated that H₂ activates calcium-related channels and promotes cytoskeletal remodeling and cell migration.

This study identifies H₂ as a novel gaseous signaling molecule that can regulate Ca²⁺ channels via the TRPC4-TRPC4AP axis. The 730Arg-731Arg motif in TRPC4 serves as a critical H₂-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic framework for developing gas-controlled H₂ regenerative therapeutics.