The transient receptor potential melastatin 8 (TRPM8) ion channel is a physiological sensor of environmental cold temperatures. This channel is also activated by menthol, which is responsible for the cooling sensation evoked by this compound. It is well known that we adapt to moderately cold temperatures, i.e. the same temperature feels less cold over time, and the cooling effects of menthol also wear off with time, presumably due to the calcium–dependent desensitization of TRPM8 activity. The membrane phospholipid phosphatidylinositol 4,5–bisphosphate (PIP2) is known to be required for TRPM8 activity. Our data support a model for desensitization, in which calcium influx through TRPM8 activates a phospholipase C enzyme, which breaks down PIP2, leading to decreased channel activity.
The activity of the cold– and menthol–activated transient receptor potential melastatin 8 (TRPM8) channels diminishes over time in the presence of extracellular Ca2+, a phenomenon referred to as desensitization or adaptation. Here we show that activation of TRPM8 by cold or menthol evokes a decrease in cellular phosphatidylinositol 4,5–bisphosphate [PtdIns(4,5)P2] levels. The decrease in PtdIns(4,5)P2 levels was accompanied by increased inositol 1,4,5 trisphosphate (InsP3) production, and was inhibited by loading the cells with the Ca2+ chelator BAPTA–AM, showing that it was the consequence of the activation of phospholipase C (PLC) by increased intracellular Ca2+ concentrations. PtdIns(4,5)P2 hydrolysis showed excellent temporal correlation with current desensitization in simultaneous patch clamp and fluorescence–based PtdIns(4,5)P2 level measurements. Intracellular dialysis of PtdIns(4,5)P2 inhibited desensitization both in native neuronal and recombinant TRPM8 channels. PtdIns(4)P, the precursor of PtdIns(4,5)P2, did not inhibit desensitization, consistent with its minimal effect in excised patches. Omission of MgATP from the intracellular solution accelerated desensitization, and MgATP reactivated TRPM8 channels in excised patches in a phosphatidylinositol 4–kinase (PI4K)–dependent manner. PLC–independent depletion of PtdIns(4,5)P2 using a voltage–sensitive phosphatase (ci–VSP) inhibited TRPM8 currents, and omission of ATP from the intracellular solution inhibited recovery from this inhibition. Inhibitors of PKC had no effect on the kinetics of desensitization. We conclude that Ca2+ influx through TRPM8 activates a Ca2+–sensitive PLC isoform, and the resulting depletion of PtdIns(4,5)P2 plays a major role in desensitization of both cold and menthol responses.