Electrophysiological recordings in HEK293 cells and [Ca2+]i changes in single COS-7 cells expressing TRPM8 channels. Cells were also transfected with the membrane-targeted FRB-CFP plasmid and either the mRFP-FKBP−fused isolated 5-ptase domain or mRFP-FKBP only. (A) Current recording measured in the whole-cell configuration, using the ramp protocol described in Materials and methods. The currents at +100 (top curves) and −100 mV (bottom curves) are shown. The averaged responses to rapa from 14 and 4 recordings for 5-ptase and FKBP-only expressing cells, respectively, measured at +100 mV are shown in the bottom histogram. (B) Averaged [Ca2+]i responses from cells expressing the mRFP−FKBP− 5-ptase domain (left, red) or mRFP-FKBP only (left, black) domain (means ± SEM from 79 and 76 cells, respectively). Representative single-cell [Ca2+]i responses from cells expressing the mRFP−FKBP−5-ptase domain are shown at the right (means ± SEM from 79 and 76 cells, respectively). Elimination of PtdIns(4,5)P
2 rapidly inhibits the whole cell current and reduces the Ca2+ signal stimulated by 500 μM menthol.