Phosphatidylinositol 4,5-Bisphosphate (PIP2) Controls Magnesium Gatekeeper TRPM6 Activity

Abstract

Magnesium (Mg2+) is crucial for cellular functions. The Mg2+-permeable channel-kinase TRPM6 controls Mg2+ homeostasis in humans, as shown by mutations of TRPM6 that cause familial hypomagnesemia with secondary hypocalcemia (HSH). TRPM6 is a member of the melastatin-related subfamily of transient receptor potential (TRP) channels. In contrast to its well-known role in human Mg2+-homeostasis, the gating mechanism of TRPM6 controlling Mg2+ influx has remained unknown. Here we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is required for TRPM6 activity and Mg2+ influx through TRPM6. Stimulation of the phospholipase-C (PLC)-coupled M1 receptor or pharmacological depletion of PIP2 potently inactivates TRPM6. Translocation of over-expressed 5-phosphatase to the 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. Activation of PLC to deplete PIP2 not only inhibits TRPM6, but also eliminates TRPM6-mediated Mg2+-influx. Furthermore, we find that the basic residues in the TRP domain of TRPM6 are putative PIP2 binding sites. Neutralization of these residues results in dysfunctional TRPM6. Similarly, these conserved residues are also crucial to TRPM7 channel function. Interestingly, we find that TRPM6 and TRPM7 preferentially bind to different PLC isoforms. However, binding to PLC is not required for PLC-induced inactivation of TRPM6 and TRPM7. Taken together, our data indicate that PIP2 is vital to TRPM6 channel function, and that hydrolysis of PIP2 by PLC-coupled hormones/agonists may constitute an important pathway for TRPM6 gating, and perhaps Mg2+ homeostasis.