Hydrogen-Induced Calcium Influx via the TRPC4-TRPC4AP Axis
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Background
Calcium ions (Ca²⁺) serve as universal intracellular messengers regulating diverse physiological processes, while dysregulated Ca²⁺ homeostasis triggers cytotoxicity. Molecular hydrogen (H₂) exhibits protective effects against oxidative stress-related pathologies, but its mechanism of action remains incompletely understood. Transient receptor potential canonical 4 (TRPC4) channels and their associated protein TRPC4AP are critical mediators of Ca²⁺ influx ( [Ca²⁺]i), yet their role in H₂-mediated calcium signaling is unexplored. This study investigates the molecular mechanism by which H₂ modulates Ca²⁺ dynamics through the TRPC4-TRPC4AP axis, aiming to establish its therapeutic potential for calcium-related disorders.
Methods
The study employed heterogeneous cellular models (e.g., mesenchymal stem cells, neurons, fibroblasts) and in vivo two-photon calcium imaging in C57BL/6J mice. Techniques included CRISPR-Cas9 knockout, siRNA-mediated gene silencing, molecular docking (AlphaFold 3), and protein-protein interaction analysis. Calcium flux was quantified via fluorescence imaging, while mitochondrial integrity and cytoskeletal dynamics were assessed using JC-1 staining, ATPase activity assays, and live-cell imaging. Structural validation of TRPC4-TRPC4AP binding sites utilized mutagenesis and complementation experiments.
Results
H₂ selectively enhanced extracellular Ca²⁺ influx via TRPC4-TRPC4AP, with no cytotoxicity or mitochondrial dysfunction observed. Key arginine residues (730Arg-731Arg) in the TRPC4 CIRB domain formed hydrogen-bond networks essential for channel activation. In vivo, H₂ increased neuronal Ca²⁺ transient frequency and amplitude in the primary motor cortex. TRPC4AP knockout abolished H₂-induced Ca²⁺ influx, while mutagenesis of 730Arg/731Arg disrupted channel activity. H₂ also promoted cytoskeletal remodeling and cell motility, dependent on TRPC4AP-mediated Ca²⁺ signaling.
Conclusions
This study identifies H₂ as a novel calcium agonist that activates the TRPC4-TRPC4AP axis to regulate extracellular Ca²⁺ influx. The 730Arg-731Arg motif in TRPC4 serves as a critical H₂-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic basis for H₂-based therapies targeting calcium dysregulation in neurodegenerative, inflammatory, and metabolic diseases, while highlighting TRPC4AP as a pivotal molecular switch for gasotransmitter signaling.
