Regulation of the TRPM3 Channel in Planar Lipid Bilayers

Abstract

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective cation channel that can be activated with nifedipine and pregnenolone sulfate (PS). The role of TRPM3 in nociceptive neurons has been linked to the heat sensation and development of heat hyperalgesia during inflammation. However, the functional characterization of TRPM3 and its specific agonists remain poorly understood. To investigate biophysical and pharmacological properties of TRPM3 we aimed to incorporate the purified channel in planar lipid bilayers. TRPM3 was purified from HEK-293 cells using immunoprecipitation technique. The single channel activity was then examined under various conditions. We investigated TRPM3 activity with different agonists. Application of nifedipine (1-10 µM) resulted in dose-dependent increase of open probability of the channel. These results suggest that nifedipine can alone activate TRPM3. Unlike nifedipine, addition to the bilayers of PS, did not induce the channel openings alone and required co-application of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or clotrimazole. TRPM3 demonstrated outward rectification upon activation with PS/PI(4,5)P2, but linear current-voltage relationship with PS/clotrimazole. In both conditions, TRPM3 tended to inactivate at higher voltages (100-120 mV) and with increased concentrations of PS. Interestingly, in the absence of any agonists TRPM3 exhibited basal channel activity that sustained for several minutes. Previously TRPM3 was proposed to serve as a temperature sensor. We tested temperature sensitivity of TRPM3 and found that in the framework of lipid bilayers the channel did not exhibit any temperature-induced activation in the absence of agonists. In the presence of nifedipine TRPM3 demonstrated weak temperature dependence with Q10of 1.5 at +100 mV and 1.7 at −100 mV. These results indicate that TRPM3 is unlikely to serve as a temperature sensor alone and may involve some alternative molecular mechanisms for temperature sensation.