Ipulations that lower plasmalemmal PIP2 in a PLCindependent manner reduce TRPM8 responsiveness to menthol. One particular caveat for this approach is that phosphataseexpressing cells are operating with lowered PIP2 levels for extended periods of time, thus potentially altering other cell functions that may perhaps indirectly affect TRPM8. Hence, we measured TRPM8 currents in cells in which we conditionally depleted plasmalemmal PIP2 levels (37), an method that has been reported to inhibit mentholevoked currents in Chlorfenapyr Protocol TRPM8expressing heterologous cells (38). This system is advantageous since it provides a method to inducibly bring the PIP2 5 phosphatase Inp54p to the cell membrane by the addition with the dimerizing immunosuppressant rapamycin. The addition of rapamycin dimerizes the phosphatase FKBPInp54p (expressed as fusion protein together with the FK506binding protein (FKBP) plus the fluorophore mCherry) using a membraneanchored FKBPrapamycinbinding domainFIGURE 5. PLCindependent depletion of plasmalemmal PIP2 reduces mentholevoked TRPM8 currents. A, representative pictures of HEK293T cells expressing rTRPM8 and LynPHPPGFP. A, panel i, GFP fluorescence marks the cells expressing each constructs and have lowered PIP2 levels. Pseudocolored images on the 340/380 nm Fura2 ratio show low basal Ca2 just before application of 200 M menthol (A, panel ii). Green arrowheads mark GFP cells expressing LynPHPPGFP in which menthol evoked a compact raise in R values, and red arrowheads mark GFPnegative cells in which menthol evoked a robust transform in intracellular Ca2 (A, panel iii). B, averaged alterations in the Fura2 ratio of manage TRPM8expressing cells (black boxes, n 20 cells) versus those coexpressing LynPHPPGFP (blue circles, n 25 cells). C, average peak ratio values (1st menthol application) of person cells and information are averaged responses from four independent experiments and 155 cells per experiment. D, representative wholecell voltage clamp recording (holding prospective (h.p.) 80 mV) from a cell transfected with rTRPM8, FKBPInp54p, and Lyn11FRB. Mentholevoked (200 M) TRPM8 currents have been diminished following application of the dimerizing agent rapamycin that translocates Inp54p to the membrane. E, wholecell voltage clamp recording from a cell transfected with rTRPM8 and FKBPInp54p but not the membrane tethered element Lyn11FRB. Repeated mentholevoked (200 M) TRPM8 currents didn’t diminish upon application in the dimerizer rapamycin. F, summary data of the reductions in mentholevoked TRPM8 currents utilizing the rapamycin (Rap), Inp54p translocation method (n five cells for every situation). G, menthol doseresponse partnership just before and following rapamycininduced Inp54p translocation and reduction of TRPM8 currents (n three cells per menthol concentration).(Lyn11FRB), thereby translocating the phosphatase for the membrane. We located that addition of 1 M rapamycin reduced wholecell mentholevoked TRPM8 currents when FKBPInp54p and Lyn11FRB have been coexpressed with TRPM8 (Fig. 5D). These data are constant with our Ca2 microfluorimetry results using the expression of membranebound Inp54p. At positive potentials, menthol currents had been lowered to 36.8 11.3 (Fig. 5F, n 5) of their original magnitude, though at adverse potentials, rapamycin proficiently eliminated TRPMVOLUME 284 Number 3 JANUARY 16,1576 1,10-Phenanthroline Technical Information JOURNAL OF BIOLOGICAL CHEMISTRYTRPM8 Is Regulated by Phospholipase C via PIPcurrents such that it excluded further evaluation (see supplemental Fig. 2B). In addition, this impact was dependent upon.