Dose-response relationships were performed and by fitting the data with a sigmoidal Hill equation we observed an IC50 of 0.6 nM at +80 mV and 0.4 nM at 280 mV . The Hill coefficient, n, was measured to be 1.1 and 1.4 at +80 mV and 280 mV, respectively, suggesting a single binding site. This makes PBMC the most potent TRPM8 antagonist reported to date . Moreover, the effects of PBMC were irreversible with no recovery of menthol-evoked currents even after a twenty minute washout . TRPM8 gating is weakly voltage-dependent in that agonists such as menthol and cold shift channel activation properties towards more negative membrane potentials, thereby facilitating channel opening at physiological voltages . Previously we found that calcium- and PIP2-dependent adaptation also correlates with a shift in TRPM8 voltage-dependence, but towards more positive membrane voltages, thereby reducing channel gating . Therefore we asked whether PBMC��s effects on menthol-evoked TRPM8 conductances produce a similar shift in the voltage dependence of the channel. In heterologous cells expressing mTRPM8, steady-state menthol-evoked currents were recorded at 23uC during voltage steps from a holding potential of 0 mV under basal Torin 1 conditions, in the presence of 1 mM menthol, or with 1 mM menthol and a near-IC50 concentration of PBMC . Normalized TRPM8 conductances for each cell, referred to as G/Gmax, were plotted for the given voltages under the three experimental conditions. We found that application of menthol alone shifted the activation curve towards negative membrane potentials, as reported previously . However, in the presence of PBMC, the menthol-induced normalized conductance shifted back towards the basal state and more positive membrane potentials . The conductance-to-voltage relationship was fitted with a Boltzmann function and we calculated the half-maximal activation voltage under each condition. The average basal V1/2 was 218.2618.2 mV, with the addition of menthol shifting this value to 79.1621.1 mV , data consistent with previous reports . When 0.5 nM PBMC was added to the bath with menthol, this shifted the average V1/2 to 171.2616.0 mV . These results suggest that PBMC PDE inhibitor antagonizes TRPM8 activity by shifting the voltage-dependence of channel gating toward more positive voltages, thereby partially reversing the effects of agonist activation. Our in vitro data show that PBMC is a profoundly potent TRPM8 antagonist with sub-nanomolar affinity. Therefore we next determined if this compound is equally effective in blocking channel function in vivo. It has been previously reported that TRP channel antagonists can affect thermoregulatory processes, most notably the undesired hyperthermic effect seen with TRPV1 antagonism . Similarly, the potent TRPM8 agonist icilin is well known to produce an intense behavioral response in rodents that is manifested as shivering, ����wet-dog���� shaking and also results in an increase in core body temperature in rats .