The planar lipid bilayer experiment is one of the unique electrophysiological methods to characterize single ion channel molecules because this technique studies specific purified ion channel proteins in the well-controlled artificial environment. Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel activated by nifedipine, neurosteroid, and temperature increase. TRPM3 is expressed in various tissues, including pancreas and central and peripheral nervous systems, and thought to be involved in insulin secretion and heat sensation. However, the functional characteristics of TRPM3 channels and its regulation by agonists still remain poorly understood. To investigate the single channel properties of TRPM3, we aimed to incorporate the purified TRPM3 protein in planar lipid bilayers and examined the single channel activity under various conditions. Application of pregnenolone sulfate alone did not induce the channel openings and co-application of the phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) was required. TRPM3 channel currents demonstrated outward rectification upon activation with pregnenolone sulfate and PI(4,5)P2. On the other hand, application of nifedipine resulted in dose-dependent increase in open probability with linear current-voltage relationship. These results suggest that activation mechanisms of TRPM3 by pregnenolone sulfate and nifedipine is different. In addition, we found that the channel did not exhibit strong temperature dependence. Increase in temperature up to 42 °C did not induce the channel openings in the absence of other agonists. While TRPM3 currents demonstrated weak temperature dependence in the presence of nifedipine. These results indicate that TRPM3 is unlikely to represent a temperature sensor by itself and some alternative molecular mechanisms may be involved in the temperature dependence.
This work was done by collaboration with Dr. Eleonora Zakharian from University of Illinois College of Medicine Peoria. This research was supported by Young Researcher Overseas Visit Program Expenses from SOKENDAI (The Graduate University for Advanced Studies).
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