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

Abstract

This study investigated the molecular basis by which H2 gas modulates intracellular calcium dynamics. Real-time calcium imaging combined with CRISPR-Cas9 knockout models demonstrated that H2-induced calcium transients were abolished in cells lacking either TRPC4 or TRPC4AP, establishing both proteins as essential mediators. Two-photon in vivo imaging in C57BL/6 mice expressing genetically encoded calcium sensors confirmed that H2 inhalation elevated calcium signals in the motor cortex and dorsal skin. Protein docking and molecular dynamics simulations identified a dual-arginine cluster (Arg730/Arg731) within the CIRB domain of TRPC4 as the H2-sensitive site; alanine substitution at these positions completely eliminated the response. H2 exposure triggered proton efflux and intracellular alkalinization, which in turn modulated the binding affinity between TRPC4 and TRPC4AP. Functionally, H2-evoked calcium transients promoted cytoskeletal remodeling and enhanced cell motility in wound-healing assays. Transcriptomic profiling corroborated activation of calcium-related channels and migration-associated gene networks, providing a comprehensive mechanistic framework for H2 as a gaseous calcium-signaling modulator.

Mechanism

H2 induces proton efflux and intracellular alkalinization, which alters the binding force between the Arg730/Arg731 motif in the CIRB domain of TRPC4 and TRPC4AP, thereby opening the TRPC4 channel and triggering extracellular calcium influx without cytotoxic overload.