Cedures”). As shown in Fig. 7B, all isozymes are present in entire TG tissue samples, whereas PLC three and PLC 4 predominate in TRPM8 3 isozymes were detected in neurons (n three experiments). C, wholecell current clamp recordings from a TRPM8expressing neuron in which trains of action potentials were elicited by two successive cold pulses (n five). D, wholecell guys samples of entire trigeminal ganglia, tholevoked (200 M) currents from TRPM8expressing neurons do not adapt to repeated stimuli in the although in 2 of 3 experiments only absence of external Ca2 and with five mM EGTA inside the recording pipette (n five). E, within the presence of physiological (2 mM) calcium and weak intracellular Ca2 buffering (0.five mM EGTA), wholecell menthol PLC 3 and 4 were observed in evoked neuronal currents adapt over time and don’t recover fully on subsequent menthol exposures purified TRPM8 neurons. Hence, when the cell is held at 22 (n 6). F, mentholevoked currents in TRPM8 TG neurons decrease or adapt these data demonstrate that upon bath application of 5 M m3M3FBS (n 7). G, currentvoltage relations at the points indicated in F. 2 H, average residual TRPM8 currents in neurons soon after application of 5 M m3M3FBS for 3 min. at each TRPM8 neurons express Ca senpositive and damaging potentials. m3M3FBS reduces currents to 66.7 17.9 and 31.2 14.three (n 7) at sitive PLC isozymes. constructive and negative membrane potentials, respectively. We subsequent characterized cold re6G, the temperature dependence of coldevoked currents sponsiveness of GFP cells electrophysiologically first in curbefore and just after Inp54p translocation was largely un rent clamp mode to identify regardless of whether action potentials were changed. Nevertheless, normalized currents at near threshold evoked in these cells by cold stimuli (a cold ramp from 40 to temperatures had been slightly diminished right after phosphatase 15 ; Fig. 7C). GFP neurons had an typical resting memtranslocation (at 24 , 0.30 0.04 before and 0.15 0.04 brane prospective of 51.six four.eight mV (n 5), and began to depoafter translocation; at 22 , 0.45 0.04 just before and 0.29 larize when the perfusate was cooled under 28.three 1.4 . Cold0.05 after translocation, p 0.05 (n 7); Fig. 6G). Temper evoked action possible amplitudes have been 76.4 eight.five mV with atureresponse profiles have been finest fit having a sigmoidal rela durations at takeoff voltage of 5.1 0.8 ms and soon after hyperpotionship (Fig. 6G), allowing for the calculation of the average larizations of four.9 1.7 mV. We observed the first action potentemperature at 20, 50, and 80 of the peak currents (at tials at an average threshold temperature of 24.2 1.six , with 14 ). Making use of these analyses (Fig. 6H), we located limited but a array of thresholds from 27.three to 18 . These data are consistsignificant differences amongst prior to and right after Inp54p ent with nerve recordings employing the skinnerve preparation in translocation in the 20 value (24.eight 0.3 before and TRPM8null mice that lack cold responses more than a array of tem22.two 0.5 after translocation, p 0.05 (n 7)). Nonetheless, peratures (9),3 suggesting that the TRPM8 population of neuno difference in temperature sensitivity was observed at rons is responsive to each innocuous and noxious cold temperother points along the temperatureresponse curve (Fig. 6H), atures in vitro. demonstrating no overt alter in channel sensitivity to cold 3 right after PIP2 LS-102 supplier depletion. C. Stucky, private communication.1578 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 284 Quantity 3 JANUARY 16,TRPM8 Is Regulated by Phospholipase C through PIPTh.
