TRPM3 Exhibits Slight Temperature Sensitivity in the Planar Lipid Bilayer System and Requires the Presence of PIP2

Abstract

Transient Receptor Potential Melastatin 3 (TRPM3) channel is critical for various physiological processes. In sensory nervous system TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, while in pancreatic β-cells the channel was linked to glucose-induced insulin release. In cells, TRPM3 is activated by heat and chemical agonists, including neurosteroid, pregnenolone sulfate (PS), and L-type Ca2+ channel blocker, nifedipine. To define the physical interactions and nuances of TRPM3 channel activity with its modulators, we succeeded to incorporate purified TRPM3 protein into the artificial membrane system - planar lipid bilayers. We found that channel opening by PS required the presence of phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole. In the presence of 5 μM PIP2 the channel exhibited EC50 for PS of ∼1.3 μM, which is lower than that observed in cell recordings (∼23 μM), due to higher agonist's sensitivity of the channel in bilayers. Unlike PS, the presence of nifedipine alone sufficed to induce TRPM3 activity (EC50 = ∼1.7 μM), in which case channel demonstrated distinct gating behavior. Similarly to PS, EC50 of TRPM3 to nifedipine in cell recordings was much higher (∼32 μM), than that observed in the bilayers.We also performed an extensive thermodynamic analysis on TRPM3 temperature dependence. Intriguingly, we found that TRPM3 exhibits slight temperature sensitivity in comparison to highly temperature-sensitive TRPM8 or TRPV1. TRPM3 remained closed upon heat-induced stimuli, but exhibited openings in the presence of PIP2 (Q10 = 5.3), although only with low open probability profile (Po = 0.048 ± 0.016).Together our results resolved the details on TRPM3 channel function in isolated system, which confirmed its direct gating by PS and PIP2, but indicated lack of strong intrinsic temperature sensitivity, common to other thermo-sensitive TRP channels.