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. 2013 Jan 18;7(2):115–118. doi: 10.4161/chan.23466

Differential inhibition of the TRPM8 ion channel by Gαq and Gα11

Lin Li 1, Xuming Zhang 1,*
PMCID: PMC3667880  PMID: 23334401

Abstract

Cold temperature is encoded by the cold-sensitive ion channel TRPM8 in somatosensory neurons. It has been unclear how TRPM8 is modulated so that it can mediate distinct type of cold signaling. We have recently reported that activated Gαq directly inhibits TRPM8 after activation of Gq-coupled receptors. Here, we further show that activation of the muscarinic receptor M1R, which is known to inhibit M currents through PLCβ-mediated hydrolysis of PtdIns(4,5)P2, similarly inhibited TRPM8 potently, but inhibition was not prevented by the PLC inhibitor U73122. Interestingly, although Gαq and Gα11 are indistinguishable in activating PLCβ and hydrolysing PtdIns(4,5)P2, activated Gα11 inhibited TRPM8 to a lesser extent than activated Gαq. The differential TRPM8 inhibition is determined by a specific residue E197 on Gα11, because mutating this residue to the corresponding residue on Gαq restored TRPM8 inhibition to a similar degree as mediated by Gαq. These results reinforce the idea that activated Gαq directly inhibits TRPM8 independently from PtdIns(4,5)P2 hydrolysis-mediated inhibition of TRPM8.

Keywords: TRPM8, heterotrimeric G proteins, GPCR, cold, pain

Introduction

Activation of the TRPM8 ion channel triggers various distinct forms of cold signaling. TRPM8 not only mediates cold perception, thermoregulation and cold analgesia,1-4 but also causes acute cold pain and cold hypersensitivity.3-7 Despite these important functions mediated by TRPM8, it is unclear how TRPM8 is modulated under pathological conditions such as inflammation. We have recently shown that inflammatory mediators such as bradykinin and histamine, both of which act on receptors coupled to Gαq, rapidly and potently inhibit TRPM8.8 Surprisingly, we found that inhibition of TRPM8 was not mainly caused by the hydrolysis of the membrane lipid PtdIns(4,5)P2 as a result of activation of PLCβ by activated Gαq, despite the fact that PtdIns(4,5)P2 is essential for maintaining TRPM8 channel activity.9,10 Instead, we found that activated Gαq protein directly inhibits TRPM8 by forming a complex with TRPM8 independently of downstream signaling pathways.8 In this study we aim to further discriminate direct inhibition of TRPM8 by Gαq from inhibition of TRPM8 caused by hydrolysis of PtdIns(4,5)P2.

Results and Discussion

To ascertain whether direct inhibition of TRPM8 by activated Gαq is a general phenomenon for Gαq-coupled receptors, we investigated regulation of TRPM8 via the muscarinic receptor M1R, whose activation is well-known to close M currents encoded by Kv7 K+ channels by depletion of the membrane lipid PtdIns(4,5)P2 due to activation of Gαq, leading to enhanced excitability of many neurons.11,12 We transfected TRPM8 cDNA into HEK293 cells, which are reported to express endogenous M1R and M3R.13 However, cells treated with carbachol, a cholinergic agonist, exhibited neither changes in PtdIns(4,5)P2 hydrolysis (data not shown) nor effects on TRPM8 inward and outward currents (Fig. 1A and B), suggesting that endogenous muscarinic receptors are not functional in coupling to TRPM8. We then recorded TRPM8 currents in HEK293 cells overexpressing exogenous M1R. Similar to bradykinin and histamine, activation of M1R by carbachol robustly inhibited both TRPM8 inward and outward currents resulting in nearly complete inactivation of TRPM8 inward currents and 76% inhibition of outward currents (Fig. 1C). As we previously reported for TRPM8 inhibition mediated via bradkykinin or histamine, pre-treatment with the PLC inhibitor U73122 had no effect in preventing TRPM8 inhibition caused by carbachol (Fig. 1C and D), though the same stock of U73122 completely inhibited PLC-mediated hydrolysis of PtdIns(4,5)P2 as well as functional sensitization of TRPV1 induced by bradykinin, a PLC signaling pathway-dependent process.8 These data show that the PLC signaling pathway is not involved in the inhibition of TRPM8 caused by activation of M1R, which does, by contrast, inhibit M currents through the PLC-PtdIns(4,5)P2 pathway.11,12 They suggest instead that a direct inhibitory action of activated Gαq protein on TRPM8 may be responsible.

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Figure 1. Activation of the muscarinic receptor M1R inhibits TRPM8. (A) Typical inward and outward currents (at −60 and +60 mV, respectively) activated by menthol (200 μM, 5 sec) in HEK293 cells expressing TRPM8. Treatment with carbachol (CCh, 1mM) had no effect. The dotted line is zero current. (B) A summary of experiments similar to those in (A). The number of experiments is shown above in each bar. Error bars are mean ± s.e.m. NS, not significant. (C) Representative inward (−60 mV) and outward currents (+60 mV) activated by menthol (200 μM, 5 sec) in HEK293 cells expressing TRPM8 and exogenous M1R were inhibited by pre-treatment with 1mM carbachol (CCh, 1 min) applied alone or together with the PLC inhibitor U73122(2.5 μM) as indicated. The dotted line is zero current. (D) A summary of the peak currents in experiments similar to those in (C). The number of experiments is given above each bar. All data are mean ± s.e.m. ***p < 0.001.

Gq-coupled receptors couple to both Gαq and Gα11. Human Gαq and Gα11 share 90% identity in protein sequence, and are considered to be two functionally redundant proteins.14 We compared the activated Gαq Q209L mutant with the activated Gα11 Q209L mutant in their ability to hydrolyze PIP2 by monitoring translocation of the fluorescence probe Tubby-R332H-cYFP, a sensitive PIP2 reporter.15Figure 2A shows that transfected tubby probes are mainly located on the cell membrane under control condition. Co-expression of the activated Gα11 Q209L caused translocation of tubby probe similarly to that induced by the activated Gαq Q209L (Fig. 2A), indicating that Gα11 Q209L and Gαq Q209L caused PLCβ activation and PtdIns(4,5)P2 hydrolysis to a similar extent. To examine whether Gα11 can cause similar membrane PtdIns(4,5)P2 degradation in a signaling context, we performed a live imaging experiment on Gαq/11-deficient MEF cells expressing the bradykinin receptor B2R together with the EGFP-tagged PLCδ-PH domain, another sensitive reporter for PtdIns(4,5)P2 metabolism. Bradykinin caused rapid translocation of the PLCδ-PH domain in MEF cells when Gα11 was co-transfected, and translocation is indistinguishable from cells co-transfected with Gαq8 (Fig. 2B and C). Therefore, Gαq and Gα11 have the same ability to activate PLCβ and catalyze membrane PtdIns(4,5)P2, consistent with previous reports.16,17

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Figure 2. Gα11 has equivalent ability to hydrolyze the membrane lipid PtdIns(4,5)P2 as Gαq. (A) Typical images of HEK293 cells transfected with Tubby-R332H-cYFP or together with the activated Gαq Q209L or Gα11 Q209L as indicated. Scale bar is 10 microns. (B) Translocation of the PLCδ-PH domain induced by bradykinin in Gαq/11-deficient MEF cells transfected with Gα11 and the bradykinin receptor B2R. Bradykinin (1 μM) was applied at 23 sec. Scale bar is 10 microns. (C) Quantification of relative membrane fluorescence signal as a function of time in (B). The area within the red dotted circle was used to quantify cytosol fluorescence signal. Bradykinin (BK, 1 μM) was applied as indicated. The experiment was repeated four times with similar results.

We then examined the regulation of TRPM8 by Gαq and Gα11 separately. We found that overexpression of wild type Gα11 had no effect on either TRPM8 inward or outward currents (Fig. 3A and B), though wild-type Gαq was observed previously to markedly inhibit TRPM8 inward currents.8 Moreover, the activated Gα11 Q209L mutant inhibited only 46% of TRPM8 inward currents and 32% of outward currents, a much weaker inhibition than that caused by activated Gαq Q209L which inhibited 98% of TRPM8 inward currents and 68% of outward currents8 (Fig. 3A and B). These results show that although Gαq and Gα11 have equivalent ability to hydrolyze PtdIns(4,5)P2, they have differential ability to inhibit TRPM8; further supporting the proposal that activated Gαq can directly inhibit TRPM8 independently of PtdIns(4,5)P2 depletion-induced inhibition of TRPM8.

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Figure 3. Activation of Gα11 inhibits TRPM8 to lesser extent than activated Gαq. (A) Example of inward (−60 mV) and outward currents (+60 mV) activated by menthol (200 μM, 5 sec) from HEK293 cells expressing TRPM8 and different Gαq/11 mutants as indicated. The dotted line is zero current. (B) A summary of TRPM8 inward and outward currents in experiments similar to those in (A). The number of experiments is indicated above each bar. All error bars are mean ± s.e.m. **p < 0.01; ***p < 0.001; NS, not significant. (C) Sequence alignment in residues from 192 to 206 between Gα11 and Gαq. Arrow indicates a key different residue.

We have previously identified the Switch III region on Gαq as a functional TRPM8 contact region causing the inhibition of TRPM8.8 However, the Switch III region is identical between Gαq and Gα11, suggesting that there are other functional contact regions on Gαq which are altered in Gα11 leading to differential TRPM8 inhibition caused by Gα11. There are 33 amino acids differences between Gαq and Gα11. Most different residues are concentrated in the N-terminal half of the protein; only five different residues are distributed across the C-terminal part of Gαq/11 encompassing amino acids 197–359. We then made a chimera G11q by replacing the C-terminal part on Gα11 Q209L with the counterpart on Gαq Q209L. The chimera G11q now inhibited TRPM8 with a similar potency to Gαq Q209L (Fig. 3A and B). This result suggests that five different residues in the C-terminal half between Gαq and Gα11 cause the differential TRPM8 inhibition. We went on to mutate these five different residues on Gα11 to the corresponding amino acids on Gαq (Fig. 3C). The activated Gα11 E197Q, Q209L mutant completely restored TRPM8 inhibition to a similar degree as the activated Gαq Q209L or the G11q chimera (Fig. 3B). Therefore, this single residue Glu197 on Gα11 determines differential TRPM8 inhibition induced by the activation of Gα11.

Gln197 residue is located between the Switch I and the Switch II region on Gαq. The Switch I and II regions are another two most mobile regions on Gαq upon activation. Structural modeling shows that Gln197 sits on a loop region forming a different protein surface at another side of Gαq protein compared with the Switch III contact region (Fig. 4). This residue may, therefore, have a role in assisting in contact of the Switch III region on Gαq with TRPM8.

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Figure 4. Structural modeling of Gαq. On the left is a ribbon representation of Gαq structure. The Switch III region is shown in pink, and Q197 residue and side chain are shown in red. On the right is a molecular surface representation. The proposed contact regions with TRPM8 on Gαq surface are shown in pink for the Switch III region and red for Q197 residue.

Taken together, our additional data further demonstrate that activated Gαq/11 is sufficient to directly inhibit TRPM8 without the need to involve the PLC-PtdIns(4,5)P2 pathway after activation of receptors coupled to Gαq/11. Although Gαq and Gα11 are traditionally considered to be redundant proteins, Gαq has a much more prominent role in regulating TRPM8 than Gα11.

Materials and Methods

Muscarinic receptor M1R was purchased from the Missouri S&T cDNA resources center. Site-directed mutagenesis was conducted by using a Quick-change mutagenesis kit (Stratagene) in accordance with manufacturer’s instructions. All other chemicals were obtained from Sigma. Whole-cell recordings were performed in HEK293 cells expressing TRPM8 or together with cDNAs encoding different G protein mutants as described previously.8 Cells were recorded in calcium free solution containing: 140 mM NaCl, 4mM KCl, 10 mM HEPES, 1 mM MgCl2, 5 mM EGTA and 5 mM glucose, adjusted to PH 7.4 with NaOH. Pipette solution contains 140 mM KCl, 2.0 mM MgCl2, 5.0 mM EGTA and 10.0 mM HEPES, adjusted to PH 7.4 with KOH. Confocal imaging and structural modeling were performed similarly as described previously.8 Differences between groups were assessed by one-way analysis of variance with Bonferroni’s post hoc test.

Acknowledgments

This work was funded by an MRC new investigator grant (G0801387 to X.Z.). We thank Dr Roger Hardie (Department of Physiology, Development and Neuroscience, University of Cambridge) for the reading of the manuscript.

Zhang X, Mak S, Li L, Parra A, Denlinger B, Belmonte C, et al. Direct inhibition of the cold-activated TRPM8 ion channel by Gαq. Nat Cell Biol. 2012;14:851–8. doi: 10.1038/ncb2529.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

References

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