Inflammatory mediators inhibit TRPM8-dependent cold nerve fibre activity. (a) Corneal nerve terminal firing frequency in response to a cold ramp (grey bars) and a heat ramp (black bars). Bath temperature shown below. Perfusion of “inflammatory soup” (5mM BK, 100mM histamine, 10mM PGE2, 100mM 5-HT and 100mM ATP) inhibited cold response (grey arrows) but enhanced heat response (black arrows). The cold response recovered to control levels following removal of IS (not shown) but heat response remained elevated. (b) Similar experiment on single afferent nerve fibres from tongue. Vertical axis shows cumulative firing during cold ramp. Filled squares: control (n=40); open squares: tongue injected with carrageenan (n=32, see Methods).

Inflammatory mediators inhibit TRPM8 independent of downstream signalling pathways. (a) BK (1μM) inhibited TRPM8-mediated calcium response in a DRG neuron responding to menthol (100μM) and capsaicin (500nM) but not mustard oil (MO, 50μM), and thus expressing TRPM8 and TRPV1 but not TRPA1. KCl (140mM) added at end. (b) Summary of mean ratio of peak calcium responses to menthol before and after application of BK alone (bar 2), as in a, or with bisindolylmaleimide (BIM, 1μM) (bar 3). Bars 2 and 3 show only BK-responsive neurons but inhibition was also significant when BK-unresponsive cells included (Supplementary Fig. S1c). Final bar is summary of experiments with PMA (see Supplementary Fig. S1b). Number of TRPM8 positive neurons shown above each bar. (c) Ca increases elicited by menthol (100μM), and effect of BK (1μM) in a single HEK293 cell transfected with TRPM8 and B2R. Ca ionophore ionomycin (Iono,10μM) added at end to saturate Ca-dependent fluorescence. (d) Summary of results similar to those in c following treatment with: neomycin (neo, 1mM); Staurosporine (Stauro,1μM); Bisindolylmaleimide (BIM, 1μM); okadaic acid (OA, 20nM) and PMA (1μM) without BK. Final bar from cells transfected with TRPM8 and TrkA receptor, treated with NGF (100ng/ml, 10min). Number of cells given above each bar. (e) Inward and outward currents (at −60mV and +60mV) activated by menthol (200μM, 5s) in HEK cells expressing TRPM8 and B2R were inhibited by pre-treatment with 1μM BK (1min) applied alone or together with inhibitors as indicated. Dashed line denotes zero current. (f) Summary of peak currents in experiments similar to those in e following treatment with U73122 (2.5μM); N-(P-amylcinnamoyl)anthranilic acid (ACA, 10μM); pertussis toxin (PTX, 1.0μg/ml); thapsigargin (Thap, 1μM). Number of experiments given above each bar. (g, h) Example (g) and summary (h) of inward and outward currents (±60mV) in response to menthol (200μM, 5s) in HEK cells expressing TRPM8 or mutants as shown together with H1R. Histamine (His, 10μM) inhibits TRPM8 current, but little affected by 2.5μM U73122. Dashed line is zero current. Number of experiments given above each bar. All data are mean±SEM, significance compared to control. **P < 0.01; ***P < 0.001; NS, not significant.

Inhibition of TRPM8 by BK is membrane-delimited. (a) Typical cell attached recording of single channel at +60mV from HEK293 cells expressing TRPM8 and B2R. Arrow indicates addition of 1μM BK. Sections of traces shown below at a higher time resolution (see alternative scale bar on left). Mean NP_o before BK, 0.13 ± 0.0092; after BK, 0.14 ± 0.0093; difference not significant, P > 0.05. On right is summary of ratio of mean NP_o before and after vehicle solution (Con), and before and after BK from the same patches. n=5, NS, not significant. (b) Similar cell-attached recording performed at +40mV on a HEK293 cell expressing TRPV1 and B2R. Patch contains multiple channels. Mean NP_o before BK, 0.01711 ± 0.0014; after BK, 0.1866 ± 0.0243; P <0.001. On right is summary of ratio of mean NP_o before and after vehicle solution (Con) or BK in the same patches. Enhancement by BK significant, ***P<0.001, n=5. Error bars in all Figs are mean ± SEM.

Activated Gα_q inhibits TRPM8 independent of PLC pathway. (a) Example of whole cell inward and outward currents (at −60mV and +60mV) activated by menthol (200μM, 5s) from HEK293 cells expressing TRPM8 and different Gα_q mutants. Dashed line is zero current. (b) Summary of TRPM8 mediated inward and outward currents when transfected with different G protein subunits in experiments similar to those in a. “Gα_q triple mutant” denotes Gα_q Q209L/R256A/T257A. Number of experiments indicated above each bar. (c) Normalized whole cell inward currents at −60mV as a function of menthol concentration in HEK293 cells or MEF cells expressing TRPM8. Control dose-response curve in HEK293 cells (●), EC₅₀=148.8μM (n=8); cotransfection with Gα_q (▲), EC₅₀=264.4μM (n=10); with 3Gα_qiq Q209L (▼), EC₅₀=356.1μM (n=9); wild type MEF cells (□), EC₅₀=156.8μM (n=5); G_αq/11^-/- MEF cells (), EC₅₀=75.1μM (n=7). All curves show Hill equation with Hill coefficient of 1.31. (d) Representative TRPM8 currents elicited by voltage steps from −140mV to + 200mV (or to +140mV in the presence of 100 μM menthol) in 20mV increments in HEK 293 cells expressing TRPM8 and Gα proteins as indicated. Dashed line is zero current. 3Gα_qiq* denotes activated Q209L mutant. Voltage-clamp protocol shown at top. (e) Conductance-voltage relationship for cells in d fitted by a Boltzmann function with values of V_½ and steepness factor as follows: TRPM8 alone (■), 119.4mV, 36.9mV; +Gα_q (▲), 162.4mV, 35.1mV; +3Gα_qiq*(Q209L) (▼), 186.1mV, 36.3mV; +100μM menthol (□), 26.3mV, 52.1mV; +Gα_q+menthol (Δ), 71.7mV, 41.9mV; +3Gα_qiq*+menthol (), 107.3mV, 38.8mV. (f) Summary of V_½ values from experiments similar to those in d. Number of experiments given above each bar. All error bars are mean ± SEM. *P<0.05; **P<0.01; ***P <0.001.

Inflammatory mediators inhibit TRPM8 via a direct action of activated Gα_q. (a) Translocation of Tubby-cYFP-R332H induced by BK in G_αq/11^-/- MEF cells co-transfected with B2R and G proteins as indicated. 1μM BK was added at 25s. Scale bar 10μm. Profiles of intensity across cells (indicated by red line) shown inset at corner of the images. (b) Quantification of relative membrane Tubby fluorescence signal as a function of time in a. Each experiment repeated at least 4 times with similar results.(c) Typical traces of TRPM8 inward and outward currents (at −60mV and +60mV), activated by menthol (200μM, 5s) in G_αq/11^-/-MEF cells transfected with H1R and TRPM8. Currents shown before (a) and after (b) histamine (10μM, 1min). Dashed line is zero current. (d) Summary of results similar to those in c. Number of experiments shown above each bar. All data are mean ± SEM. **P<0.01; NS, not significant. (e) Similar experiments with application of BK (1μM) to G_αq/11^-/- MEF cells expressing B2R. (f) Summary of results similar to those in e. All bars are mean ± SEM. *P<0.05; ***P<0.001; NS, not significant.

Direct interaction of TRPM8 with Gα_q. (a, b) Mutual co-precipitation between TRPM8 and Gα_q. HEK293 cell lysate expressing TRPM8-V5-his and Gα_q/Gα_q Q209L was pulled down by either nickel beads (a) or Gα_q antibody (b), co-precipitated Gα_q (a) or TRPM8 (b) was detected with indicated antibodies (top blots). Same blots stripped and reprobed with anti-V5 (a) or anti-G α_q (b) (middle blots). Bottom blots show similar Gα_q (a) or TRPM8 (b) expression in total cell lysate (TCL) in all cases. (c) In DRG neurons Gα_q was co-precipitated by TRPM8. Same blot reprobed with anti-TRPM8 (bottom). Specificity shown by omission of TRPM8 antibody from IP (Con, left lane). (d) Gα_q and Gα_q Q209L bind to TRPM8 N and C terminal. GST-coupled TRPM8 N and C terminal used to pull down cell lysate expressing Gα_q or Gα_q Q209L (top blot). N terminal binds 1.28±0.025 fold more Gα_q than does C terminal (n=3). Blot stripped and reprobed with anti-GST (bottom). (e) Bradykinin and histamine did not enhance binding of Gα_q to TRPM8. Cells expressing TRPM8-V5-His and B2R or H1R were treated with DSP after exposure to bradykinin (1μM, left) or histamine (10μM, right) for different minutes. TRPM8 pulled down by Ni-NTA, and the associated Gα_q detected by anti-Gα_q (top blot). Blots reprobed with anti-V5 (bottom). (f) TRPM8 binds to switch III region on Gα_q, resulting in the suppression of TRPM8. On left is schematic diagram of chimeras between Gα_q Q209L (black bars) and Gα_i2 Q205L (grey bars). Number of amino acids in each protein used for the chimera given at the top corner of black bars (Gα_q Q209L), or underneath grey bars (Gαi2 Q205L). Switch I, II and III regions on Gα_q in purple, TRPM8 and PLCβ binding regions indicated by dotted blue lines. Ability of chimeras to activate PLCβ (Supplementary Fig. S4a) indicated to right of each chimera, +, activated; –, ineffective. Second bar from bottom shows construction of 3Gα_qiq in which distal C-terminal of G α_q (333-359) has been transplanted back into 3Gα_qi in order to allow coupling to G_q-coupled GPCRs. Right: summary of TRPM8 currents caused by chimeras shown on left. Number of experiments shown to left of each bar. Error bars, S.E.M. *P<0.05; ***P<0.001; NS, not significant. Uncropped images of blots (6a-e) shown in supplementary Fig.S8.

Activated Gα_q directly inhibits TRPM8 in excised patches. (a) Left: typical example of channel activity at +40mV in inside-out patches excised from HEK cells expressing TRPM8 after addition of 50nM Gα_q* (Gα_q pre-incubated with GTPγS) in the presence of 50μM DiC8-PIP₂. Arrows indicate time of addition of Gα_q*. Sections of traces are shown below at higher time resolution. Note that single-channel currents are smaller than in Fig. 3a, because membrane potential was lower. (b) Real time quantification of NP_o in a. Red dashed lines give mean NP_o over indicated time period. Inset shows silver stain of purified Gα_q protein. Mean NP_o before Gα_q*, 0.151 ± 0.0088; after Gα_q*, 0.013 ± 0.001; P<0.001. (c) Similar experiment to a, but with application of 100μM GTPγS. (d) Real time quantification of NP_o in c. Red dashed lines give mean NP_o over indicated time period. NP_o before GTPγS, 0.20±0.016; after GTPγS, 0.019 ± 0.0027; P<0.001; (e) Mean values of ratios of NP_o for treatments shown below bars (ratio is mean value over period 1minute after application, to that 1min before, see plots in b, d). Number of experiments given above each bar. All data are mean ± SEM. ***P<0.001 compared to control solution; NS, not significant relative to control. (f) Cartoon depicting proposed mechanism for regulation of TRPM8 by Gα_q subunit. Left: in resting state at 34°C, TRPM8 is pre-bound to Gα_q. TRPM8 activity is low at this temperature. Middle: cold temperature or a cooling compound such as menthol (M) activates TRPM8, resulting in an inward current flow and an increase in firing frequency of cold fibres. Right: Inflammatory mediators (L) released by tissue injury bind to a GPCR resulting in GTP-GDP exchange and consequent activation of Gα_q. The consequent conformational change in Gαq causes inhibition of TRPM8 by a direct interaction, resulting in a decrease in current passing into the nerve terminal through TRPM8 and so a decreased firing of cold-sensitive nerve fibres.