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

## TL;DR

This study shows that hydrogen gas triggers calcium signals in cells through a specific protein complex, offering a new understanding of its therapeutic effects.

## Contribution

The study identifies TRPC4-TRPC4AP as a molecular target for hydrogen gas in regulating calcium signaling.

## Key findings

- H2 induces reversible calcium transients in a TRPC4-TRPC4AP-dependent manner.
- H2 activates cell motility and alters intracellular pH through TRPC4-TRPC4AP interaction.
- The 730Arg-731Arg motif in TRPC4 is critical for H2-induced calcium influx.

## Abstract

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.