TRPM6 is crucial for human Mg2+ homeostasis as patients carrying TRPM6 mutations develop hypomagnesemia and secondary hypocalcemia (HSH). However, the activation mechanism of TRPM6 has remained unknown. Here we demonstrate that phosphatidylinositol-4,5-bisphophate (PIP₂) controls TRPM6 activation and Mg2+ influx. Stimulation of PLC-coupled M1-receptors to deplete PIP₂ potently inactivates TRPM6. Translocation of over-expressed 5-phosphatase to cell membrane to specifically hydrolyze PIP₂ also completely inhibits TRPM6. Moreover, depolarization-induced-activation of the voltage-sensitive-phosphatase (Ci-VSP) simultaneously depletes PIP₂ and inhibits TRPM6. PLC-activation induced PIP₂-depletion not only inhibits TRPM6, but also abolishes TRPM6-mediated Mg2+ influx. Furthermore, neutralization of basic residues in the TRP domain leads to nonfunctional or dysfunctional mutants with reduced activity by PIP₂, suggesting that they are likely to participate in interactions with PIP₂. Our data indicate that PIP₂ is required for TRPM6 channel function; hydrolysis of PIP₂ by PLC-coupled hormones/agonists may constitute an important pathway for TRPM6 gating, and perhaps Mg2+ homeostasis.

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