Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate

Abstract

Cold is detected by a small subpopulation of peripheral thermoreceptors. TRPM8, a cloned menthol- and cold-sensitive ion channel, has been suggested to mediate cold transduction in the innocuous range. The channel shows a robust response in whole-cell recordings but exhibits markedly reduced activity in excised membrane patches. Here we report that phosphatidylinositol 4,5-bisphosphate (PIP2) is an essential regulator of the channel function. The rundown of the channel is prevented by lipid phosphatase inhibitors. Application of exogenous PIP2 both activates the channel directly and restores rundown activity. Whole-cell experiments involving intracellular dialysis with polyvalent cations, inhibition of PIP2 synthesis kinases, and receptor-mediated hydrolysis of PIP2 show that PIP2 also modulates the channel activity in the intact cells. The crucial role of PIP2 on the function of TRPM8 suggests that the membrane PIP2 level may be an important regulator of cold transduction in vivo. The opposite effects of PIP2 on the vanilloid receptor TRPV1 and TRPM8 also implies that the membrane lipid may have dual actions as a bimodal switch to selectively control the heat- and cold-induced responses in nociceptors expressing both channels.

Figure 1.

Rundown of TRPM8 channels. A, Representative trace for the rundown of currents recorded from a Xenopus oocyte membrane patch. The patch was initially maintained in the cell-attached (c/a) mode and then excised into the normal bath solution. The pipette solution contained 100 μm menthol. Mg²⁺ (1.8 mm) was applied at the end to examine the extent of rundown. Currents were evoked with V_h = -60 mV at room temperature (∼22°C). B, Comparison of the time course of rundown in the normal bath solution and solutions containing Mg²⁺ and FVPP, respectively. The currents were normalized with the on-cell responses as the maximum and the residual currents after exposure to Mg²⁺ as the minimum. C, Quantification of the rundown under different conditions. The rundown rate was measured as the half-decay time relative to the initial cell-attached responses. D-F, Parallel experiments for cold responses at 10°C. i/o, Inside-out.

Figure 2.

Effects of PIP₂ on TRPM8 in excised patches. A, A monoclonal antibody (28.5 μg/ml) specific to PIP₂ inhibited channel activity. The recording was made in the FVPP bath solution with 100 μm menthol included in the pipette. B, Application of PIP₂ (20 μm) restored channel activity after rundown. The normal bath solution was used, and other conditions were the same as in A. PIP₂ was delivered to the cytoplasmic face in the FVPP solution. C, Direct activation of the channel by PIP₂ (20 μm). Before application of PIP₂, the patch was first excised into the normal bath solution to allow for rundown of a small spontaneous activity and then further exposed to the Mg²⁺-containing solution for a complete rundown. D, Half-maximal times of PIP₂-induced recovery and activation and the steady-state amplitudes of the currents restored by PIP₂ and PIP expressed as a percentage of the currents obtained before patch excision. E, Dose-response curves of menthol-activated currents recorded under the condition with and without the inclusion of 30 μm PIP₂ in the pipette solution. FVPP was present in both bath and pipette to prevent channel rundown. The solid lines represent fits to the Hill equation, with EC₅₀ = 36 μm and n = 1.3 in the absence of PIP₂ (4-10 patches) and EC₅₀ = 12 μm and n = 1.1 in the presence of PIP₂ (4-12 patches). All recordings were from Xenopus oocyte membrane patches with the inside-out (i/o) configuration for A-D and the outside-out configuration for E. Holding potential, -60 mV.

Figure 3.

Inhibition of TRPM8 by polyvalent cations. A, Intracellular dialysis of PLL (0.01%) through a patch pipette inhibited whole-cell currents induced by 100 μm menthol in HEK293 cells. The first response was recorded immediately after break-in to the whole-cell configuration. B, Summary graph of the effects of different polyvalent cations. The inhibition was determined as the percentage of peak current remaining in an interval of ∼10 min relative to the initial response after patch break-in. C, D, Inhibition of cold-induced activity of the channel. Cooling was applied by exchanging the bath content with solutions precooled to 10°C, followed by warming back to room temperature. All recordings were made at a holding potential of -60 mV.

Figure 4.

Block of PIP₂ synthesis inhibits TRPM8 activity. A, Representative whole-cell recordings showing the effects of wortmannin at 10 and 1 μm, respectively. Currents were evoked by 100 μm menthol applied externally. Wortmannin was dialyzed into the cell through the pipette solution. B, Comparison of the inhibition induced by wortmannin at 10 and 1 μm and by PAO at 10 μm. The percentage of current remaining at the end of ∼10 min application of the blockers relative to the first response was plotted. PAO was applied by extracellular perfusion. C, D, Effects of wortmannin and PAO on temperature responses. Currents were activated by cooling bath to 10°C as described previously. E, Current trace from an outside-out patch elicited by 100 μm menthol in the absence and presence of wortmannin and PAO, showing no direct effects of the inhibitors on the channel. FVPP was included in both bath and pipette solutions to prevent channel rundown. F, Summary graph for experiments in E showing the percentage currents in the presence of the inhibitors relative to the currents obtained with 100 μm menthol alone. Data were recorded at -60 mV from HEK293 cells transiently expressing TRPM8.

Figure 5.

Inhibition of TRPM8 through trkA receptor-mediated hydrolysis of PIP₂. A, Whole-cell recordings on the effects of NGF on menthol and cold activity of the channel, respectively. NGF (5 nm) was applied externally for 10-20 min after the first recording. B, Summary graph of NGF-induced inhibition from different experiments including manipulation of each step in the signaling pathway downstream of the NGF receptor trkA. The NGF effect was evaluated as the percentage of remaining current after 10 min treatment relative to the initial response before treatment. Except explicitly noted, all experiments were performed with 100 μm menthol as the stimulus. Data were recorded at -60 mV from HEK293 cells transiently transfected with TRPM8 and the NGF receptor trkA/p75 or its mutants. TG, Thapsigargin.

Figure 6.

Inhibition of TRPM8 via muscarinic receptor-mediated hydrolysis of PIP₂. A, Whole-cell recording of menthol-activated currents from an HEK293 cell coexpressing TRPM8 and the rat M₁ receptor. Carbachol (CCh; 5 μm) was applied externally in the presence of menthol (100 μm). B, Summary plot of carbachol effects on TRPM8. The currents were normalized to the initial response of menthol before carbachol application. The control experiments corresponded to those with transfection of TRPM8 alone. All currents were elicited with 100 μm menthol at a holding potential of -60 mV.