Competitive Interactions between PIRT, the Cold Sensing Ion Channel TRPM8, and PIP₂ Suggest a Mechanism for Regulation

TRPM8 is a member of the transient receptor potential ion channel family where it functions as a cold and pain sensor in humans and other higher organisms. Previous studies show that TRPM8 requires the signaling phosphoinositide lipid PIP₂ to function. TRPM8 function is further regulated by other diverse mechanisms, including the small modulatory membrane protein PIRT (phosphoinositide regulator of TRP). Like TRPM8, PIRT also binds PIP₂ and behavioral studies have shown that PIRT is required for normal TRPM8-mediated cold-sensing. To better understand the molecular mechanism of PIRT regulation of TRPM8, solution nuclear magnetic resonance (NMR) spectroscopy was used to assign the backbone resonances of full-length human PIRT and investigate the direct binding of PIRT to PIP₂ and the human TRPM8 S1-S4 transmembrane domain. Microscale thermophoresis (MST) binding studies validate the NMR results and identify a competitive PIRT interaction between PIP₂ and the TRPM8 S1-S4 domain. Computational PIP₂ docking to a human TRPM8 comparative model was performed to help localize where PIRT may bind TRPM8. Taken together, our data suggest a mechanism where TRPM8, PIRT, and PIP₂ form a regulatory complex and PIRT modulation of TRPM8 arises, at least in part, by regulating local concentrations of PIP₂ accessible to TRPM8.