Computational and functional characterization of the PI(4,5)P2 binding site of the TRPM3 ion channel

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)P2], but the molecular mechanism of activation by PI(4,5)P2 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)P2 via molecular modeling. We identified putative contact residues for PI(4,5)P2 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)P2 levels by phosphatidylinositol 4-kinase inhibition. Changes in ligand-binding affinities via MM/GBSA showed reduced PI(4,5)P2 affinity for the mutants. Mutating PI(4,5)P2 interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate (PregS), pointing to an allosteric interaction between PI(4,5)P2 and PregS. Mutating residues in the PI(4,5)P2 binding site in TRPM8 had similar effects, increased sensitivity to PI(4,5)P2 depletion, and reduced sensitivity to menthol. Mutation of most PI(4,5)P2 interacting residues in TRPM3 also increased sensitivity to inhibition by Gβγ, indicating allosteric interaction between Gβγ and PI(4,5)P2. Disease-associated gain of function TRPM3 mutations on the other hand, resulted in no change of PI(4,5)P2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P2, its relationship to endogenous activators and inhibitors of TRPM3, as well as identify similarities and differences between PI(4,5)P2 regulation of TRPM3 and TRPM8. ### Competing Interest Statement The authors have declared no competing interest.