Rapamycin activates TRPM8 in HEK293 cells and sensory neurons.

(A) Time course of the intracellular calcium concentration in HEK293 cells expressing TRPM8, showing robust responses to rapamycin (10 µM) and menthol (50 µM), and inhibition of the responses by AMTB (2 µM). Shown are mean ± SEM, N=63 cells

(B) Concentration dependence of rapamycin-evoked calcium responses. The dashed line respresents the best fit using a Hill equation.

(C) Left, time course of whole-cell currents in HEK293 cells expressing TRPM8 evoked by repetitive voltage steps to +120 and -80 mV, showing the activation of outwardly rectifying currents by rapamycin (10 µM) and inhibition by AMTB (2 µM). Right, voltage steps recorded at the indicated time points.

(D) Concentration dependence of rapamycin-evoked whole-cell currents at +120 mV. The dashed line respresents the best fit using a Hill equation.

(E) Examples of calcium signals in individual DRG neurons from Trpm8+/+ and Trpm8-/- mice in response to rapamycin (Rapa; 10 µM), menthol (Menth; 50 µM), pregnenolone sulphate (PS; 40 µM), cinnamaldehyde (CIN; 10 µM), capsaicin (Caps; 100 nM) or high K+ (50 mM).

(F) Fraction of sensory neurons from Trpm8+/+ and Trpm8-/- mice that responded to rapamycin and menthol.

Direct interaction between rapamycin and TRPM8.

(A) Left, time course of currents in a cell-free inside-out patch pulled from a HEK293 cell expressing TRPM8 evoked by repetitive voltage steps to +80 and -80 mV, showing the activation of outwardly rectifying currents by rapamycin (10 µM) and menthol (50 µM) applied from the cytosolic side. Right, voltage steps recorded at the indicated time points. This example is representative for 5 similar experiments.

(B) Cartoon representing the different steps to obtain a STTD spectrum from rapamycin interacting with TRPM8.

(C) 1H NMR spectra for the indicated conditions.

(D) Comparison of the 1H NMR spectra of rapamycin in buffer and in the presence of TRPM8 with the resonance assignments.

(E) Structure of rapamycin; hydrogen atoms involved in the interaction with TRPM8 are indicated by the yellow circles.

TRPM8 residues involved in the interaction with rapamycin and menthol.

(A) Representative time courses of the intracellular calcium concentration in HEK293 cells expressing wild type TRPM8 or the indicated mutants, when stimulated with rapamycin (10 µM), menthol (50 µM) and the calcium ionophore ionomycin (2 µM).

(B) Quantification of the relative calcium response to rapamycin and menthol for wild type and the indicated TRPM8 mutants. Values indicate the ratio between the calcium response amplitude to rapamycin, divided by the sum of the responses to rapamycin and menthol. The dotted line represents the mean value for wild type TRPM8. Values above this line (in yellow) indicate a relative reduction in the response to menthol, whereas values below the line (cyan) indicate a relative reduction in the response to rapamycin.

(C) Amplitude of the calcium response to the agonist (menthol or rapamycin) that gave the largest response for wild type and the indicated TRPM8 mutants. Data on B and C represent mean ± SEM, N=34-156/group.

(D) Whole-cell current-voltage relations for the currents in control, and in the presence of rapamycin (10 µM) or menthol (50 µM) in HEK293 cells expressing wild type TRPM8 or the indicated mutants.

(E) Quantification of the relative current response to rapamycin and menthol for wild type and the indicated TRPM8 mutants. Values indicate the ratio between the current amplitude increase at +80 mV to rapamycin, divided by the sum of the responses to rapamycin and menthol. The dotted line represents the mean value for wild type TRPM8. Values above this line (in yellow) indicate a relative reduction in the response to menthol, whereas values below the line (cyan) indicate a relative reduction in the response to rapamycin.

Structural model showing the distinct interaction sites for menthol and rapamycin.

(A) Side view (left) and top view (right) of TRPM8, showing the known interaction site for menthol (green) and the proposed rapamycin interaction site (red) based on our present molecular docking and mutagenesis studies.

(B) Closer view of rapamycin docked onto the TRPM8 structure. Amino acid residues that, when mutated, influence rapamycin responses are indicated in green.

(C) 2D projection of interactions between rapamycin and TRPM8 created using Ligplot+.

Activation of TRPM8 by rapamycin is independent of intracellular calcium.

(A) Whole-cell currents during 800-ms voltage steps from -80 to +80 mV under control conditions and in the presence of rapamycin (10 µM) or icilin (10 µM). At the time points indicated by the arrows, a 1-ms UV falsh was applied, leading to a rapid increase in intracellular calcium. Magenta lines and scale bar indicate Fura-FF fluoresence ratios at the indicated time points.

(B) Whole-cell current-voltage relations measured during voltage ramps 2 s before and 1 second after the UV flashes shown in panel A.

(C) Current amplitudes at +80 and -80 mV before and after UV uncaging of calcium, under control conditions and in the presence of rapamycin or icilin.

(D) Quantification of the relative potentiation of inward and outward currents following UV uncaging of calcium. Data in C and D represent the mean ± SEM from 5 experiments.

Effect of rapamycin and macrolid analogs on TRPM8.

(A) Overview of the macrolids tested in this study.

(B) Relative calcium response to rapamycin and the indicated analogs tested at 10 µM. N=6 in each group.

(C) Fura2-based calcium response to rapamycin (10 µM) in the presence of everolimus (10 µM) or vehicle. N=6 in each group.

(D) Fura2-based calcium response to menthol (50 µM) in the presence of everolimus (10 µM) or vehicle. N=6 in each group.

(E) Summary of the responses to menthol (50 µM) and rapamycin (10 µM) in the absence or presence of everolimus (10 µM). Responses were normalized to the response to a saturating concentration of menthol (300 µM). N=6 in each group.

Rapamycin does not activate TRPA1, TRPM3 or TRPV1.

(A-C) Representative whole-cell current-voltage relations in CHO-cells expressing mouse TRPA1 (A), HEK293 cells expressing mouse TRPM3 (B) or HEK293 cells expressing human TRPV1 (C) upon stimulation with rapamycin (30 µM) or the respective agonists Allyl isothiocyanate (AITC), pregnenolone sulphate (PS; 40 µM) and capsaicin (Caps; 100 nM). Current responses to rapamycin where <5% of the response to the respective channel agonists in n=5 cells.

Rapamycin allows distinghuising between TRPM8-mediated and TRPM8-independent menthol responding sensory neurons.

(A) Representative examples of Fura2-based calcium signals in two sensory neurons that were stimulated three times at the indicated times with rapamycin (10 µM) or menthol (50 µM). During the second application, the TRPM8 antagonist AMTB was present at a concentration of 2µM.

(B) Average ratio between the response to menthol in the presence and absence of AMTB (second response/first response) in neurons that did or did not respond to rapamycin.

Rapamycin acts as a type I agonist.

(A) Representative examples of whole-cell currents in HEK293 cells expressing TRPM8, in response to a 200-ms voltage step from -80 to +120 mV and back to -80 mV, in control conditions and in the presence of the indicated concentrations of rapamycin or menthol.

(B) Zoomed-in time course of current relaxation at +120 mV. Dashed lines indicate monoexponential fits.

(C) Zoomed-in time course of current relaxation at -80 mV. Dashed lines indicate monoexponential fits.

(D) Monoexponential time constants for current activation at +120 mV in control conditions and in the presesence of the indicated concentrations of menthol or rapamycin.

(E) Monoexponential time constants for current deactivation at -80 mV in control conditions and in the presesence of the indicated concentrations of menthol or rapamycin. **, ***: P<0.01, P<0.001 versus control. ##, ###: P<0.01, P<0.001 versus menthol. Mean ± SD, dots represent individual cases.

Potential binding sites and poses of rapamycin and TRPM8 obtained from pilot blind dockings.

(A-B) site 1, (C-D) site 2, (E-F)site 3. Rapamycin is shown in slate color; binding site residues are colored in orange; grey dashed lines indicate intermolecular contacts; slate spheres indicate groups in direct contact with TRPM8 as indicated by STTD-NMR.

Molecular docking of rapamycin and everolimus to the groove between voltage sensor-like domain and the pore domain of TRPM8.

(A-B) In silico molecular docking indicates that everolimus binds to TRPM8 in a similar pose as rapamycin, albeit with lower binding energy (-8.5 kcal/mol for everolimus versus -11.6 kcal/mol for rapamycin).

(C-D) A zoom-in on the binding site shows the hydrogen bond between the hydroxyl group on the cyclohexane ring in rapamycin (C) and the side-chain amide of residue Gln861, whereas no such a hydrogen bond can be formed between the longer hydroxyethyl moiety on everolimus (D).

(E-F) 2D projections of interactions between rapamycin or everolimus and TRPM8 created using Ligplot.