Computational and functional characterization of the PI(4,5)P 2 binding site of the TRPM3 ion channel

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ABSTRACT 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 by phosphatidylinositol 4-kinase inhibition. Changes in ligand-binding affinities via 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 (PregS), pointing to an allosteric interaction between PI(4,5)P 2 and PregS. Mutating residues in the PI(4,5)P 2 binding site in TRPM8 had similar effects, increased sensitivity to PI(4,5)P 2 depletion, and reduced sensitivity to menthol. Mutation of most PI(4,5)P 2 interacting residues in TRPM3 also increased sensitivity to inhibition by Gβγ, indicating allosteric interaction between Gβγ and PI(4,5)P 2 . 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 of TRPM3, as well as identify similarities and differences between PI(4,5)P 2 regulation of TRPM3 and TRPM8.

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