Phosphoinositide Regulation of TRPM8 Channels in Planar Lipid Bilayers

Abstract

The cold and menthol receptor TRPM8 is regulated by membrane phosphoinositides. To study the effects of lipids directly on the channel, we have reconstituted the purified TRPM8 in planar lipid bilayers. This system allows full control of the lipid composition in our experiments. The reconstituted channel was activated by menthol or cold, and its activity depended on the presence of specific phosphoinositides. In the presence of menthol, TRPM8 exhibited the highest probability of opening in the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] with Po ∼ 0.89 at +100 mV and Po ∼ 0.4 at −100 mV. Less channel activity was induced by phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] with Po ∼ 0.53 at +100 mV and Po ∼ 0.2 at −100 mV. Phosphatidylinositol 3,4,5-bisphosphate [PI(3,4,5) P2] resulted in irregular TRPM8 channel currents and lower open probability with Po ∼ 0.21 at +100 mV and Po ∼ 0.087 at −100 mV. Among the tested lipids the lowest TRPM8 channel activity was induced by phosphatidylinositol 4-phosphate [PI(4)P] with Po ∼ 0.12 at +100 mV and Po ∼ 0.019 at −100 mV. The lipid specificity profile in lipid bilayers is very similar to that observed in excised patches.We have also studied the activation of TRPM8 channels in lipid bilayer with cold. Cooling the system with reconstituted TRPM8 channels also required the presence of PI(4,5)P2. The main shift in the channel behavior was observed in the temperature range from 21°C to 18°C where the channel showed drastic changes in the open probability from 0.05 to 0.85 at +100 mV.These results demonstrate that the TRPM8 protein is directly activated by cold, menthol and phosphoinositides.