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 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.
Highlights
PIRT is a phosphoinositide binding membrane protein with two transmembrane (TM) helices and intracellular N- and C-termini
HPIRT was eventually reconstituted in DPC (n-dodecylphosphocholine) for future studies. human PIRT (hPIRT) is stable in DPC for weeks and gives relatively well-resolved spectra, for a helical membrane protein
We have previously shown that hPIRT directly binds the human TRPM8 (hTRPM8)-S1S4 domain with a stoichiometry of ~1:112
Summary
PIRT is a phosphoinositide binding membrane protein with two transmembrane (TM) helices and intracellular N- and C-termini It is expressed primarily in the dorsal root and trigeminal ganglia of the peripheral nervous system[22] and appears to modulate thermosensing through apparent interactions with the cold-sensing TRPM812–14 and the heat-sensing TRPV1 ion channels[12,22,23,24]. To probe the molecular mechanism of human TRPM8 (hTRPM8) modulation by PIRT, we used solution nuclear magnetic resonance (NMR) spectroscopy, microscale thermophoresis (MST), and Rosetta computational techniques to isolate how human PIRT (hPIRT), PIP2, and hTRPM8 interact. We computationally docked PIP2 to the hTRPM8-TMD to illuminate the potential location of the TRPM8–PIRT–PIP2 ternary complex From these studies, we propose a mechanism where hPIRT exerts at least partial modulatory control of hTRPM8 by regulating PIP2 accessibility
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