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Activation of the TRPM8 ion channel in sensory nerve endings produces a sensation of pleasant coolness. Here we show that inflammatory mediators such as bradykinin and histamine inhibit TRPM8 in intact sensory nerves, but do not do so via conventional signalling pathways. The G-protein subunit Gaq instead binds to TRPM8 and when activated by a Gq-coupled receptor directly inhibits ion channel activity. Deletion of Gaq largely abolished inhibition of TRPM8, and inhibition was rescued by a Gaq chimera whose ability to activate downstream signalling pathways was completely ablated. Activated Gaq protein, but not Gβγ, potently inhibits TRPM8 in excised patches. We conclude that Gaq pre-forms a complex with TRPM8 and inhibits activation of TRPM8, following activation of G-protein coupled receptors, by a direct action. This signalling mechanism may underlie the abnormal cold sensation caused by inflammation.
The temperature sensitive ion channels TRPV1 and TRPM8 play an essential role in the pain pathway. Inflammatory mediators released during tissue injury or inflammation enhance heat pain by sensitizing the heat-gated TRPV1 ion channel via intracellular signalling pathways whose endpoint is phosphorylation of TRPV1 by protein kinases1-5. The TRPM8 ion channel is activated by cold temperatures6, 7, and is also involved in many aspects of pain sensation such as cold analgesia, and paradoxically cold hypersensitivity8-12. Acute activation of TRPM8, by cooling or by application of agonists of TRPM8 such as menthol, causes an analgesic effect8, 9. Cold hypersensitivity, on the other hand, is observed in chronic inflammatory conditions, and increased TRPM8 expression appears to be an underlying mechanism8, 10, 13. It is thus unclear how TRPM8-expressing cold thermoreceptors may be affected by inflammation.
The membrane PIP2 level reulates TRPM8 activity. TRPM8 activity increased when PIP2 was applied to the intracellular surface of excised patches14, 15, and was reduced when membrane PIP2 was depleted16. A decrease in membrane PIP2, caused by activation of the PLC pathway following binding of an agonist such as bradykinin (BK) to a Gq-coupled GPCR, was therefore assumed to be responsible for inhibiting TRPM8 channel activity14, 15. However, other studies have proposed that PKC is instead involved in the inhibition of TRPM8 by BK17, 18.
In the present study we examined the effect of inflammatory mediators on TRPM8-dependent nerve activity, and we confirm that TRPM8 is inhibited by inflammatory mediators which couple to Gαq. However, we found that the inhibition of TRPM8 activity is largely independent of cell signalling pathways downstream of Gαq-coupled receptors. Neither depletion of membrane PIP2 nor phosphorylation by PKC is crucial. Instead we found that activated Gαq binds to TRPM8, and causes inhibition by a direct action following activation of Gαq-coupled receptors.
Cold-sensitive fibres innervating the cornea of the eye detect small changes in ambient cold temperature, a property solely dependent on TRPM8 channel expression19. We first examined the effect of inflammatory mediators on cold-sensitive fibres of the cornea. A cooling ramp from 34°C to 20°C elicited an increasing discharge of nerve impulses (Fig. 1a), known to be attributable to activation of TRPM819. A heat ramp to 48°C initially suppressed ongoing TRPM8-dependent activity, but then reactivated firing at 45°C, likely because of heat–dependent activation of TRPV1. However, when an “inflammatory soup” (IS), containing BK and histamine was perfused, increases in firing frequency evoked by cooling were smaller (grey arrows in Fig. 1a, mean peak frequency before IS, 44.3±4.5 impulses per second; after IS, 31.8±3.4; n=12, P<0.001, paired t test). A significantly larger temperature decrease (ΔT) for initiation of increased firing was also observed (ΔT before IS, 1.1±0.4°C; after IS, 1.8±0.4°C; n=12, P<0.01, paired t test). In contrast, the firing frequency evoked by heat was enhanced by inflammatory mediators (black arrows, Fig. 1a). We found no significant desensitization of firing frequency under control condition when saline solution was perfused (mean peak frequency before saline solution, 47.5±5.95 impulses per second; after saline, 44.0±7.7; n=4, P>0.05, paired t test; Supplementary Fig. S1a)19. These data show that inflammatory agents suppress TRPM8-mediated responses to cooling in intact cold thermoreceptor terminals in situ, while at the same time enhancing heat responses.
A similar result in cold-sensitive afferent nerve fibres of the mouse tongue was obtained when inflammation was induced by carrageenan injection. Fig. 1b shows the cumulative sum of firing in single sensory afferent nerves as the temperature was lowered from 34°C to 12°C; the integral of firing as a function of temperature was half-activated at 31.6±1.09°C (n=40) in control fibres but at 25.4 ±0.81°C (n=32) in inflamed fibres.
We next measured the activity of TRPM8 in cultured DRG neurons by applying pulses of menthol, an agonist for TRPM8, and monitoring the rise in [Ca]i caused by channel activation. In agreement with nerve fibre recordings, BK potently inhibited TRPM8 in 11 out of 33 TRPM8 positive neurons (Fig. 2a). PKC-mediated phosphorylation, either direct or via activation of a phosphatase, has been suggested to be responsible for inhibition of TRPM817, 18, but we found that the BK-induced inhibition of TRPM8 was not abolished by the PKC inhibitor BIM, and nor was it mimicked by the PKC activator PMA (Fig. 2b; Supplementary Fig. S1b,c). Similar observations were made in HEK293 cells co-transfected with TRPM8 and the BK receptor B2R, in which BK inhibited TRPM8 with a similar efficacy to DRG neurons (Fig. 2c,d; Supplementary Fig. S1d). B2R-dependent inhibition of TRPM8 was not affected by the specific PKC inhibitor BIM, nor by the broad PKC inhibitor staurosporine, the phosphatase inhibitor okadaic acid, nor the PLC inhibitor neomycin (Fig. 2d). Moreover, activation of PKC by PMA did not cause significant inhibition of TRPM8 (Fig. 2d), though PMA had a marked effect on TRPV1 (Supplementary Fig. S1e). These experiments do not favour the idea that signalling pathways downstream of PLC may underlie the effect of BK on TRPM8.
To extend these experiments, the current flowing through TRPM8 channels was monitored during voltage-clamp pulses to ±60mV or in full I-V curves (Supplementary Fig. S2a,b), and the effects of inhibitors on signalling pathways were investigated. Membrane PIP2 is known to activate TRPM814, 15, and therefore PIP2 hydrolysis following activation of PLCβ by Gq-coupled GPCRs could be a mechanism for inhibiting TRPM8. This idea is not supported, however by the inability of U73122, a PLC inhibitor, to prevent the inhibition of TRPM8 currents (either inward or outward) caused by BK or histamine (Fig. 2e-h). The same concentration of U73122 completely inhibited PLC-mediated hydrolysis of PIP2 and also inhibited the sensitization of TRPV1 induced by BK (Supplementary Fig. S3a,b), a process dependent on the PLC signalling pathway5, 20.
Moreover, histamine strongly inhibited TRPM8 currents in two PIP2-insensitive TRPM8 mutants, K995Q and R1008Q14 (Fig. 2g, h). We also found that activation of PLCγ via application of NGF had no inhibitory effect on TRPM8 (Fig. 2d, last bar). These experiments suggest that receptor-mediated hydrolysis of PIP2 is not sufficient to inhibit TRPM8. A possible pathway involving activation of PLA2 followed by coupling to Gαi is also not supported by the lack of effect of the PLA2 inhibitor ACA and inactivation of Gαi/o by PTX (Fig. 2e,f). Disruption of intracellular Ca2+ signalling by applying the Ca uptake inhibitor thapsigargin, by buffering intracellular calcium with BAPTA-AM or by blocking the IP3 receptor with 2-APB also had no effect on BK-induced inhibition of TRPM8 currents, suggesting that intracellular Ca2+ release is not involved (Fig. 2f; Supplementary Fig. S1f).
Taken together, these data indicate that the conventional intracellular signalling pathways downstream of PLC are not involved in TRPM8 inhibition, and we therefore investigated other possible mechanisms.
Whether a diffusible intracellular mediator is involved in the inhibition of TRPM8 by BK can be determined by making cell-attached patch recordings of single channels and applying BK only outside the patch. Sensitization of TRPV1 depends on activation of kinases by the PLC signalling pathway5, and as expected application of BK outside the patch potently enhanced channel activity (Fig. 3b). TRPM8 single channel bursting, by contrast, was not inhibited by bath application of BK (Fig. 3a). These experiments suggest that BK-induced inhibition of TRPM8 is membrane-delimited and depends on local events within the patch, and not on diffusible messengers.
The local nature of TRPM8 channel inhibition suggested that activated Gαq itself may cause direct inhibition of TRPM8, as previously suggested for K channels21, 22. Gαq has two forms, an inactive GDP-bound and an active GTP-bound form. Over-expression of Gαq had a small inhibitory effect on TRPM8 inward current (Fig. 4a), presumably because a small proportion of Gαq is in the active GTP-bound form even in the absence of GPCR stimulation23. The Gαq/11 Q209L mutant, which is deficient in intrinsic GTPase activity and is therefore mainly in the GTP-bound active configuration24, caused a much greater inhibition of both inward and outward TRPM8 currents (Fig. 4a,b).
Inhibition of TRPM8 by active Gαq could result from potent activation of PLCβ, and consequent hydrolysis of PIP2. To test this possibility, we used a sensitive reporter of membrane PIP2 levels, Tubby-cYFP-R332H25, to monitor activation of the PLCβ/PIP2 pathway. As expected, expression of the active mutant Gαq Q209L, which couples to PLCβ, caused complete translocation of Tubby to the cytoplasm, while wild type Gαq, which is largely in the inactive GDP-bound form, had no detectable effect (Supplementary Fig. S4a). A commonly used triple mutant Gαq Q209L/R256A/T257A, which has been reported to be unable to activate PLCβ26, profoundly suppressed TRPM8 currents (Fig. 4b), but we found this mutant still caused substantial Tubby translocation (Supplementary Fig. S4a), and so in fact still couples to PLCβ. We therefore constructed a chimera between Gαq and Gαi2 by replacing the PLCβ binding region on Gαq, by the corresponding region on Gαi2 (Fig. 6f). We found that this chimera, which we named 3Gαqiq (see below), completely failed to deplete PIP2 even when made constitutively active by the Q209L mutation (Supplementary Fig. S4a). However, when activated by the Q209L mutation, 3Gαqiq strongly inhibited both inward and outward TRPM8 currents (Fig. 4a, b), showing that the 3Gαqiq chimera retains the ability to couple to TRPM8, even though its coupling to PLCβ is selectively disabled. The inhibition of TRPM8 was specific to Gαq, because other activated Gα subunits (Gαi2 Q205L and Gα13 Q226L) and Gβ1γ2 were without effect (Fig. 4b). Collectively, these experiments show that activated Gαq can directly inhibit TRPM8, independent of downstream PLC pathway.
The effect of Gαq on TRPM8 inward currents activated by menthol is shown in Fig. 4c. Over-expression of Gαq reduced TRPM8 sensitivity to menthol, because a small fraction is in the active GTP-bound conformation (see above). The constitutively active but signalling-ablated mutant 3G αqiq Q209L caused a stronger inhibition of TRPM8 sensitivity to menthol. Conversely, expression of TRPM8 in MEF cells lacking endogenous Gα 27 q/11 , enhanced TRPM8 sensitivity to menthol, showing that endogenous Gαq imposes a tonic inhibition on TRPM8. Outward TRPM8 currents can be evoked by strong depolarization in either the absence or presence of menthol, and these currents were suppressed by Gαq and to an even greater extent by 3Gαqiq Q209L, leading to a shift of the G-V curve and a significant positive shift in V½ (Fig. 4d-f). We noticed that currents recorded in cells without menthol consistently have a larger noise than in the presence of menthol, presumably caused by a flickering opening of TRPM8 channels (see below Fig. 7a,b). These results show that TRPM8 activation, whether by menthol or by depolarization, is inhibited by active Gαq by shifting the voltage dependence of TRPM8 towards more positive membrane potentials. The strong inhibition caused by the signalling-ablated mutant 3Gαqiq Q209L shows that inhibition occurs without engagement of downstream signalling pathways.
To further investigate whether the coupling of the 3Gαqiq chimera to PLCβ is completely disabled, we transfected the PIP2 reporter Tubby-R332H-YFP along with the BK receptor into Gαq/11-null MEF cells. Tubby rapidly translocated to the cytoplasm following BK treatment when wild type Gαq was co-transfected, but there was no translocation with the 3Gαqiq chimera (Fig. 5a,b). Similar results were obtained with another PLCβ signalling reporter PLCδ-PH-EGFP (Supplementary Fig. S4b,c). These data indicate that the 3Gαqiq chimera lacks the ability to activate PLCβ.
We then used the 3Gαqiq chimera in gain-of-function experiments in the Gαq/11-deficient MEF cells. Histamine caused no suppression of TRPM8 activity, confirming the complete deletion of Gαq/11 in the MEF cells, but transfection of the 3Gαqiq chimera rescued suppression (Fig. 5c, d; Supplementary Fig. S2d). Similarly, the 3Gαqiq chimera also rescued BK-mediated inhibition of TRPM8 currents (Fig. 5e, f; Supplementary Fig. S2c). The rescue of coupling from GPCRs to TRPM8 by 3Gαqiq, which is unable to couple to PLCβ, confirms that Gαq couples directly to TRPM8 without the need to involve signalling pathways downstream of PLC.
Direct modulation of TRPM8 by Gαq suggests that they might form a complex. Fig. 6a shows that both wild-type Gαq and active Gαq Q209L were pulled down by TRPM8 to a similar extent. Reciprocally, TRPM8 was co-precipitated by either Gαq or Gαq Q209L (Fig. 6b). Co-precipitation between TRPM8 and Gαq was also observed in native DRG neurons (Fig. 6c). Both the N- and C-terminal domains of TRPM8 bind to Gαq and to Gαq Q209L, though stronger binding was observed to the N terminus (Fig. 6d). Neither Gαi2 nor Gαs showed significant binding to TRPM8 under similar conditions (Supplementary Fig. S5a-c). Furthermore, neither BK nor histamine promoted binding of Gαq to TRPM8 (Fig. 6e). Thus Gαq and TRPM8 form a constitutive complex, and activation of Gαq by a GPCR does not enhance binding.
We next delineated the functional TRPM8 binding region on Gαq by making a series of chimeras between active forms of Gαq, which binds to and activates TRPM8, and Gαi, which does not (shown schematically in Fig. 6f). All chimeras were similarly expressed, and none affected TRPM8 expression (Supplementary Fig. S5d,e). Chimeras Gαqi and 2Gαqi activated PIP2 hydrolysis in a similar manner to Gαq Q209L. However, 3Gαqi lacked the ability to hydrolyse PIP2 (Supplementary Fig. S4a), showing that the PLCβ binding region on Gαq is located between E245 and Y261, in agreement with other findings26. Interestingly, this chimera still inhibited TRPM8 inward and outward currents, but a progressive loss of inhibition was found in chimeras 4Gαqi and 5Gαqi (Fig. 6f), and a corresponding loss of binding to TRPM8 was observed with the same deletions (Supplementary Fig. S5d), indicating that the region between N221 and N245 on Gαq contains the functional TRPM8 binding region required for the modulation of TRPM8. This region corresponds to the Switch III helix region of Gαq, which is structurally one of the most mobile regions and has extensive contacts with effectors. A modelled structure of heterotrimeric Gαqβγ indicates that the Switch III loop protrudes out of the protein surface and is free to be engaged by effectors such as TRPM8 (Supplementary Fig. S6). TRPM8 and PLCβ therefore bind to distinct but contiguous regions on Gαq, rendering their mutual and independent regulation by Gαq possible.
If activated Gαq protein inhibits TRPM8 without the intervention of downstream signalling pathways, then the inhibition should be detectable in excised TRPM8-containing patches. TRPM8 channel activity runs down immediately after excision due to rapid loss of PIP214, 15, but channels then remain active at a constant low level. Subsequent application of a water-soluble DiC8-PIP2 restored channel activity to an even higher level (Supplementary Fig. S7a). We applied purified Gαq protein, activated by prior incubation with GTPγS, to the intracellular surface of excised patches when channel run down was complete and activity had become stable. Activated Gαq rapidly reduced the TRPM8 open probability (Supplementary Fig. S7b), an effect which could be due to activation of residual PLCβ trapped in the patch, and a consequent further reduction in levels of PIP2. We found, however, that the inhibition of TRPM8 by activated Gαq was even more prominent in the presence of saturating levels of DiC8-PIP2, which strongly activates TRPM8 (Fig. 7a,b). In control experiments, application of Gαq incubated with GDPβS, which forces Gαq into the inactive state, of boiled Gαq, of Gβγ, or of the activation buffer without GTPγS or Gαq, were all without effect (Fig. 7e).
We showed above that Gαq constitutively binds to TRPM8. Endogenous Gαq should therefore remain in excised patches, associated with TRPM8, and should inhibit TRPM8 when switched into an active state. Consistent with this idea, addition of non-hydrolysable GTPγS, but not GDPβS, to inside-out membrane patches reduced the TRPM8 open probability both in the presence or absence of exogenous DiC8-PIP2 (Fig. 7c-e; Supplementary Fig. S7c). In experiments on patches excised from MEF cells lacking endogenous Gαq/11 the application of activated Gαq itself inhibited TRPM8 as in patches from HEK293 cells, but the inhibitory effect of GTPγS alone was absent (Fig. 7e), confirming that Gαq remaining in the patch is indeed responsible for TRPM8 inhibition. This experiment also shows that subunits other than Gαq/11 are not able to inhibit TRPM8 following activation by GTPγS.
Three mechanisms for modulation of ion channels by GPCRs have been well established. Channels can be modulated by phosphorylation by kinases such as PKA, PKC or Src28, 29, by direct binding to Gβγ subunits released following GPCR activation30, 31, or by interaction with membrane PIP232-35. We provide here evidence for a fourth mechanism: the Gαq subunit binds directly to TRPM8, and the conformational change of Gαq following GPCR activation causes a rapid and direct trans-inhibition of TRPM8 (Fig. 7f). It is tempting to speculate that the Gαq subunit could be involved in the regulation of other ion channels, a possibility that can now be investigated by the use of our 3Gαqiq chimera, which allows unequivocal discrimination of a direct action of activated Gαq from downstream actions triggered by the PLC signalling pathway.
Previous studies have demonstrated that PIP2 is a potent activator of TRPM814-16. A decrease in membrane PIP2 following activation of a Gq-coupled GPCR could therefore be responsible for inhibiting TRPM8. In the present study we confirmed that activation of Gq-coupled GPCRs caused a marked PIP2 depletion. Unexpectedly, our evidence argues against a major physiological role for PIP2 depletion in TRPM8 inhibition, because inhibiting PLC had little effect on TRPM8 inhibition following activation of Gq-coupled GPCRs, whereas a Gαq construct completely decoupled from PLC still had a strong action. Therefore, direct inhibition by activated Gαq causes the major part of the inhibition. A likely explanation for the lack of involvement of PIP2 is that following activation of a Gq-coupled GPCR, TRPM8 is rapidly engaged and inhibited by activated Gαq even before the downstream PLCβ signalling pathway is initiated, so that subsequent PIP2 depletion cannot further inhibit the channels. Another possibility is that the binding affinity of TRPM8 channels for PIP2 is high, so that channels remain fully occupied by PIP2 even when PIP2 have been depleted by activation of PLC32, 35.
We found that Gαq binds to TRPM8 even when inactive, and that binding was not enhanced by activation of Gαq. It is likely that inactive Gαq binds to TRPM8 at an interface which differs from that of activated Gαq, and that a conformational change upon activation causes a reorientation of Gαq to interact with a different site on TRPM8. We base this proposal on the observation that in isolated membrane patches, application of activated Gαq is able to inhibit TRPM8, even though TRPM8 is already bound to endogenous inactive Gαq. Gαq thus functions as an integral component of the TRPM8 gating machinery, controlling the opening of TRPM8 channels and allowing rapid and efficient signal transduction. A similar association is also observed between G αq and PLCβ, which form a preassembled complex36, and in which activation of Gαq does not increase the association with PLCβ37.
A recent study has also reported an interaction between TRPM8 and Gαq38. This study showed that activation of TRPM8 causes downstream activation of the Gαq-PLC metabotropic pathway. This effect is in the reverse direction to the Gαq-to-TRPM8 inhibition demonstrated in the present paper, and raises the possibility that not only can Gαq influence the gating of TRPM8, as shown here, but that the gating of TRPM8 may also influence Gαq. Activation of TRPM8 could in this scenario activate Gαq, which would then inhibit TRPM8. In the absence of external Ca2+, however, there is little rebound inhibition of TRPM8 currents following activation by menthol (see for example Fig. 4a), suggesting that any activation of Gαq by TRPM8 plays only a minor role in the gating of TRPM8.
The sensory information provided by cold-sensitive receptors is involved in the conscious sensation of coolness, in the detection of skin-surface dryness and in cold allodynia11, 19. The evidence that inflammatory mediators can inhibit the activity of cold-sensitive nerve terminals by a direct action of Gαq on TRPM8 has relevance for the understanding of cold disesthesias associated with injury and inflammation. Elucidating the molecular mechanism involved in the modulation of cold-evoked activity under inflammatory conditions opens up new possibilities for its selective therapeutic manipulation.
Corneal nerve fibre recording was performed as described previously19. Briefly, eyes of adult C57BL/6J mice were removed and placed in a recording chamber perfused with the saline solution. A fire-polished glass recording pipette filled with physiological saline was applied to the surface of the corneal epithelium with slight suction to make extracellular recordings of nerve activity. Signals were amplified with an AC amplifier and data were captured and analyzed using a CED 1401 interface coupled to a computer running Spike2 6.0 software.
Tongue nerve fibre recording was performed in isolated tongues from adult male C57BL/6J mice. The tongue was removed from the head and right and left lingual nerves were isolated. The tongue was then transferred to a recording chamber continuously perfused with saline solution at 35°C. The distal end of one of the lingual nerves was placed in an adjoining compartment filled with paraffin oil and split into smaller filaments. A filament was placed on a monopolar platinum wire electrode connected to an amplifier to record impulse activity. When a filament displaying spontaneous activity was detected, a cold ramp down to around 10°C was delivered. Nerve filaments showing multiunit background discharge with cooling were further divided until a nerve filament containing a single or few active units was obtained. To induce an acute inflammation in the tongue, animals were injected with lambda carrageenan (2% in saline, 5μl) down the midline towards the tip of the underside of the tongue. This caused a marked tissue edema that was fully developed two hours later. The mouse was then sacrificed, the tongue was prepared and recorded in the same manner as above.
HEK 293 cells and MEF cells were maintained in DMEM medium containing 10% fetal bovine serum, 100 UI/ml penicillin, 100 μg/ml streptomycin and 2.0 mM L-Glutamine. For electrophysiology, HEK 293 cells were transiently transfected using polyfect transfection reagents (QIAGEN) according to manufacturer’s instructions. MEF cells were transfected by using cell line nucleofection kits (Lonza). Electrophysiology recordings were typically performed 2~3 days following transfection. For molecular biology experiments, we use TurboFect transfection reagent (Fermentas).
cDNA constructs: Human Gαq/11, Gαi2, Gβ1, Gγ2, Gαs, Gα13 and H1R receptor cDNAs were purchased from Missouri S&T cDNA Rescource Center. GST coupled TRPM8 N and C terminal fragments and all Gαq chimers were constructed by standard PCR procedures. TRPM8 tagged with V5 and hexahis epitopes at C terminal were described as previously5. Mutagenesis was performed by using Quick-Change site-directed mutagenesis kit (Stratagene).
Pull down assays and co-immunopreciptation: To pull down hexahistidine-tagged TRPM8 with nickel beads, HEK293 cells transfected with TRPM8-V5-His and Gαq were solubilised in a lysis buffer consisting of 20mM HEPES, 1.0% NP40, 150mM NaCl, 0.4mM EDTA and 20mM imidazole plus protease inhibitor cocktail (Roche). Ni-NTA agarose beads (QIAGEN) were then incubated with cell lysate at 4°C followed by extensive wash with the lysis buffer. For GST pull down, c. 0.2μg purified GST coupled N (1~691) and C terminal (980~1104) protein fragments from BL-21 cells were incubated with either purified Gαq protein or cell lysate overexpressing G αq/Gαq Q209L at 4°C for 3 hours, followed by overnight incubation with GST-agarose and centrifugation. For cross-linking experiments in Fig. 6e, treated HEK293 cells were incubated with 2.0mM cell permeable DSP (Dithiobis[succinimidyl propionate], Pierce) cross linker for 30 minutes before solubilisation and co-immunoprecipitation. All washed beads were boiled in sample buffer and loaded on 10% SDS-PAGE gel for western blot analysis. Coimmunoprecipittion was performed as described previously4, 5. For co-immunoprecipitation from DRG neurons in Fig. 6c, TRPM8 antibody (Transgenic Inc, Japan, KM060, 1:100) was used to precipitate TRPM8 in DRG neurons, and associated Gαq was detected by monoclonal anti-Gαq (Santa Cruz, sc-136181, 1:1000). Polyclonal anti-Gαq antibody (Santa Cruz, sc-393, 1: 2000) which recognizes Gαq N terminal domain was used for the detection of Gαq and all chimeric Gαq proteins. Antibodies against Gαi2 (sc-13534, 1:2000) and Gαs (sc-46975, 1:2000) were from Santa Cruz. All blots repeated at least three times with similar results.
Electrophysiological experiments were performed in calcium free bath solution unless otherwise stated to prevent desensitization of TRPM8.
Whole cell patch recording was performed largely as described previously5. Briefly, patch electrodes were pulled from thin walled glass capillaries, and had a resistance of 3.0~4.0MΩ when filled with internal solution with the following composition (in mM): 140 KCl, 2.0 MgCl2, 5.0 EGTA, 10 HEPES, PH 7.4 with KOH. Cells were perfused with calcium free bath solution containing (in mM): 140 NaCl, 4 KCl, 10 HEPES, 1 MgCl2, 5 EGTA, 5 glucose, pH 7.4 with NaOH. For experiments using calcium containing bath solution, EGTA was replaced with 1.8mM CaCl2. TRPM8 inward and outward currents were measured at a holding potential of −60mV and +60mV, respectively. Cells were pre-treated with 1μM bradykinin or 10μM histamine for 1minute before break-in to the whole cell mode to measure TRPM8 currents activated by menthol. To examine TRPM8 activation by depolarization, steps of voltage pulses were applied for 100ms ranging from −140mV to +200mV in 20mV increments, followed by a final step to +60mV. Half maximal activation voltage (V1/2) was obtained as described previously5 by fitting normalized channel conductance (G/Gmax)-voltage relationship to a Boltzmann equation: G/Gmax=1/(1+exp[-(Vm-V1/2)/k]). All recordings were made at room temperature (24°C) with an Axopatch 200B patch clamp amplifier (Axon) in conjunction with pClampex 10.2 version software (Molecular Devices). Signals were analog filtered using a 1 kHz low-pass Bessel filter.
Cell attached and inside-out recordings were made using pipettes fabricated from thick wall borosilicate glass tubing (Sutter Instrument) with a resistance of 9~15 MΩ when filled with pipette solution. Pipettes were fire polished using a microforge and coated with Sigmacote(Sigma). For inside-out recordings we used a pipette solution with the following composition (in mM):140 NaCl, 3 KCl, 10 HEPES, pH7.3 with NaOH. Bath solution contained (in mM): 140 KCl, 5 EGTA, 1 MgCl2, 10 HEPES, 5 glucose PH7.3 with KOH. Menthol (500μM) was included in the pipette solution to activate TRPM8 channels within the patch. Recordings were sampled at 5 kHz and filtered at 2.0 kHz. For experiments in Fig. 7a, b, 50μM DiC8-PIP2 (Echelon Biosciences) was present in the bath solution to prevent TRPM8 channel run-down. Activated G proteins were pulsed onto the excised patches through an ejection pipette positioned close to the patches. Ejection pipettes were connected to a PicoSpritzer III ejection system using nitrogen as a pressure source. Single channel data were analyzed using Clampfit10.2 software (Molecular Devices). Overall channel activities of patches (NPo) were obtained by using the “50% threshold criterion” from the idealized traces39. All events were carefully checked visually before being accepted. For representation purposes traces were filtered at 500Hz.
Gαq protein was expressed and purified as described40. Briefly, human Gαq, Gβ1 and hexahis tagged Gγ2 were subcloned into transfer vector PVL1392. Each was cotransfected into Sf9 cells together with baculovirus flashback GOLD expression vector, and recombinant baculoviruses containing G protein subunits were amplified. Sf9 cells were then infected with a combination of those baculoviruses at a M.O.I of 3.0. Cells were harvested 48h after infection and solubilised by 1% sodium cholate. Proteins were purified by Ni-NTA agarose column followed by extensive washing. Gαq subunit was eluted from the column by 30μM AlCl3 and subsequently purified by HiTrap Q HP anion exchange column (GE Healthcare). Proteins were eluted with a gradient of NaCl, peak fractions were collected and assayed by immunoblot with anti-Gαq antibody. Fractions containing the Gαq protein were pooled and concentrated to 0.3mg/ml using an Amicon Ultra filter and stored in aliquot of 3.0 μl at −80°C.
Gαq protein aliquots were activated in the presence of 0.2mM DTT, 1mM GTPγS and 0.01% CHAPS in the patch bath solution at 30°C for 50 minutes. To remove excess GTPγs after activation of Gαq, the buffer for activated Gαq protein was exchanged by repeated dilution with bath solution and centrifugation by using an AmiconUltra fiter. Similar procedures were followed for deactivating Gαq with GTPβS.
We also purchased purified human Gαq protein from Origene, both sources of purified Gαq protein showed a similar effect. Purified bovine brain Gβγ subunits were obtained from Merck Biosciences.
Calcium imaging was performed at room temperature as described previously4. Briefly, transfected HEK293 cells or DRG neurons were plated onto a coverslip and loaded with Fluo-4-AM (Invitrogen). Cells were continuously perfused with normal Hanks solution and images were collected every 3s using a Bio-Rad confocal microscope. Pulses of menthol (100μM) were applied for 15 seconds every 4 minutes. Bradykinin (BK, 1μM) was applied for 2 minutes between the 5th and 6th menthol response. The effect of BK was quantified as a response ratio by dividing the 6th by the 5th peak response amplitude. In control experiments on cells not exposed to BK the distribution of response ratios was found to be well fitted by a normal distribution (supplementary Fig. S1d), from which a threshold ratio was derived at 95% confidence level and used to determine cells significantly inhibited by BK.
Tubby-R332H-cYFP and PLCδ-PH-EGFP translocation was determined by live-scanning using a Leica confocal microscopy. Images of MEF cells transfected with the fluorescence probe, B2R and G proteins as appropriate were collected every 0.75s. Probe translocation was quantified by calculating the ratio of membrane fluorescence to that of cytosol using ImageJ software.
All data are mean ± SEM. Difference between groups were assessed by either paired (Fig. 1a, 3a,b) or unpaired Student’s t test (Fig. 5d, f and Supplementary Fig S3b), or by one way analysis of variance (ANOVA) with Bonferroni’s post hoc test (for all other figures). Results were considered significant at P<0.05.
We thank Dr. S. Offermanns for providing MEF cells, Dr. G. Hammond for Tubby-R332H and PLCδ-PH-EGFP cDNA and for help with imaging analysis, Dr. O. Opaleye for help with protein purification, Dr. R. Hardie and Dr. S.B. Hladky for help with single channel recording, and Dr. R. Hardie for critical reading of an earlier version of the manuscript. This work was supported by an MRC new investigator research grant (G0801387 to X.Z.), a BBSRC research grant (BB/F003072/1to P.A.M.) and a grant from the BBVA (to P.A.M. ., to support a BBVA visiting professorship at the Instituto de Neurociencias, Alicante).
AUTHOR CONTRIBUTIONS X.Z. came up with the hypothesis, designed and performed experiments, and analysed data except calcium imaging experiments, which were carried out by S.M., and nerve fibre recordings which were carried out by A.P, B.D., C.B. and P.A.M.; L.L. assisted with molecular biology experiments and neurons preparation. X.Z. wrote the manuscript with input from C.B. and P.A.M. X.Z. and P.A.M. supervised the project.