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

Abstract

Abstract 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 PIP 2 to function. TRPM8 function is further regulated by other diverse mechanisms, including the small modulatory membrane protein PIRT ( p hospho i nositide r egulator of T RP). Like TRPM8, PIRT also binds PIP 2 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 PIP 2 and the human TRPM8 S1-S4 transmembrane domain. Microscale thermophoresis (MST) binding studies validate the NMR results and identify a competitive PIRT interaction between PIP 2 and the TRPM8 S1-S4 domain. Computational PIP 2 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 PIP 2 form a regulatory complex and PIRT modulation of TRPM8 arises, at least in part, by regulating local concentrations of PIP 2 accessible to TRPM8.