# Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx.
> 分子状水素はTRPC4-TRPC4AP軸を介して細胞外カルシウム流入による可逆的カルシウム過渡応答を誘発する


## 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.

## Bibliographic

- **Authors**: Zhao PL, Li H, Cai ZY, Zhang XQ, Wen X, Liu Z, et al.
- **Journal**: Theranostics
- **Year**: 2026
- **PMID**: [41799190](https://pubmed.ncbi.nlm.nih.gov/41799190/)
- **DOI**: [10.7150/thno.124352](https://doi.org/10.7150/thno.124352)
- **PMC**: [PMC12964245](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12964245/)
- **Study type**: in vitro study
- **Delivery route**: inhalation
- **Effect reported**: positive

## Delivery context

For inhalation applications of molecular hydrogen, the lower flammability limit (LFL) deserves careful handling. The classical 4% figure applies to closed-system mixtures; the practical inhalation-environment threshold is 10%. Even pure-hydrogen output (the UFL 75% paradox) passes through the flammable range at the air–gas boundary. High-concentration (66% / 100%) inhalers are documented in the Japanese Consumer Affairs Agency accident-information database and are not recommended.

## Safety notes

For inhalation applications of molecular hydrogen, the lower flammability limit (LFL) deserves careful handling. The classical 4% figure applies to closed-system mixtures; the practical inhalation-environment threshold is 10%. Even pure-hydrogen output (the UFL 75% paradox) passes through the flammable range at the air–gas boundary. High-concentration (66% / 100%) inhalers are documented in the Japanese Consumer Affairs Agency accident-information database and are not recommended.

See also:
- [Inhalation concentration and LFL / UFL](https://h2-papers.org/en/safety-notes/inhalation-concentration)
- [Consumer Affairs Agency accident cases](https://h2-papers.org/en/safety-notes/accident-cases)
- [LFL / UFL terminology](https://h2-papers.org/en/safety-notes/lfl-ufl-explained)
- [Inhalation safety threshold lineage](https://h2-papers.org/en/safety-notes/lineage)

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> **Cite as**: H2 Papers — PMID 41799190. https://h2-papers.org/en/papers/41799190
> **Source**: PubMed PMID [41799190](https://pubmed.ncbi.nlm.nih.gov/41799190/)