Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2), but the molecular mechanism of activation by PI(4,5)P-2 is not known. As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PI(4,5)P-2 via molecular modeling. We identified putative contact residues for PI(4,5)P-2 in the pre-S1 segment, the S4-S5 linker, and the proximal C-terminal TRP domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PI(4,5)P-2 levels. Changes in ligand-binding affinities via molecular mechanics/generalized Born surface area (MM/GBSA) showed reduced PI(4,5)P-2 affinity for the mutants. Mutating PI(4,5)P-2-interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate, pointing to an allosteric interaction between PI(4,5)P-2 and pregnenolone sulfate. Similarly, mutating residues in the PI(4,5)P-2 binding site in TRPM8 resulted in increased sensitivity to PI(4,5)P-2 depletion and reduced sensitivity to menthol. Mutations of most PI(4,5)P-2-interacting residues in TRPM3 also increased sensitivity to inhibition by G beta gamma, indicating allosteric interaction between G beta gamma and PI(4,5)P-2 regulation. Disease-associated gain-of-function TRPM3 mutations on the other hand resulted in no change of PI(4,5)P-2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P-2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P-2, its relationship to endogenous activators and inhibitors, as well as identify similarities and differences between PI(4,5)P-2 regulation of TRPM3 and TRPM8.