The transient receptor potential channel TRPM8 is inhibited via the alpha 2A adrenoreceptor signaling pathway

Abstract

The transient receptor potential channel melastatin member 8 (TRPM8) is expressed in sensory neurons, where it constitutes the main receptor of environmental innocuous cold (10-25 degrees C). Among several types of G protein-coupled receptors expressed in sensory neurons, G(i)-coupled alpha 2A-adrenoreceptor (alpha 2A-AR), is known to be involved in thermoregulation; however, the underlying molecular mechanisms remain poorly understood. Here we demonstrated that stimulation of alpha 2A-AR inhibited TRPM8 in sensory neurons from rat dorsal root ganglia (DRG). In addition, using specific pharmacological and molecular tools combined with patch-clamp current recordings, we found that in heterologously expressed HEK-293 (human embryonic kidney) cells, TRPM8 channel is inhibited by the G(i) protein/adenylate cyclase (AC)/cAMP/protein kinase A (PKA) signaling cascade. We further identified the TRPM8 S9 and T17 as two key PKA phosphorylation sites regulating TRPM8 channel activity. We therefore propose that inhibition of TRPM8 through the alpha 2A-AR signaling cascade could constitute a new mechanism of modulation of thermosensation in both physiological and pathological conditions.

FIGURE 1.

Co-expression of α2A-AR with TRPM8 channels in HEK-293 cells induces inhibition of TRPM8-carried current (I_TRPM8) by the α2A-adrenergic agonist, clonidine. A, representative time courses of I_TRPM8 (measured as current density at +100 mV) in response to TRPM8-activating stimuli (shown by horizontal bars): temperature drop from 33 to 20 °C (cold), icilin (10 μm), and menthol (500 μm) in HEK-293_M8 cells transiently transfected with the α2A subtype of α2-adrenoreceptor (HEK-293_M8-α2A-AR) under control conditions (black circles) or following treatment with clonidine (10 μm, open circles). B, I-V plot of A. C, quantification of the inhibitory effect of clonidine in cold-, icilin-, and menthol-activated I_TRPM8 in HEK-293_M8-α2A-AR cells (mean ± S.E., n = 5–10 for each cell type and condition). Each TRPM8-activating stimulus was applied independently of each other. (**) and (***) denote statistically significant differences with p < 0.02 and p < 0.01, respectively.

FIGURE 2.

Functional link between α2A-AR and TRPM8 involves G_i proteins, but not G_q proteins and PLC. A, quantification of the effects of HEK-293_M8 cell dialysis with the constitutive G protein activator, GTPγ-S (100 μm, white columns) and inhibitor, GDP-β-S (100 μm, gray columns), on icilin- and menthol-activated I_TRPM8 (mean ± S.E., n = 6–10 for each cell type and condition). B, average ramp-derived I-V relationships of menthol-activated I_TRPM8 in HEK-293_M8 cells dialyzed with GTPγ-S-free (black triangles) and GTPγ-S-containing pipette solution with (black circles) and without (open circles) cell pretreatment with PLC inhibitor, U73122 (1 μm). The inset shows quantification of I_TRPM8 densities at + 100 mV under respective conditions (mean ± S.E., n = 6–10 for each condition). C, quantification of the inhibitory effect of clonidine on the density of cold-, icilin-, and menthol-activated I_TRPM8 in HEK-293_M8-α2A-AR cells pretreated (gray columns) and not pretreated (open columns) with U73122 (mean ± S.E., n = 6–10 for each condition). D, quantification of the effects of HEK-293_M8 cell transient transfection with the wild-type (WT, light gray column), constitutively activated (Q205L, open column) or constitutively inactivated (G204A, gray column) forms of the G_i α-subunit on menthol-activated I_TRPM8 densities; black column represents HEK-293_M8 blank plasmid-transfected control (mean ± S.E., n = 6–10 for each cell type). E, quantification of the effects of clonidine on the density of cold-, icilin-, and menthol-activated I_TRPM8 in HEK-293_M8-α2A-AR cells pretreated (gray columns) and not pretreated (open columns) with G_i inhibitor, PTX (500 ng/ml, mean ± S.E., n = 6–10 for each condition). On all graphs (*) and (**) denote statistically significant differences to control or between connected values with p < 0.05 and p < 0.02, respectively.

FIGURE 3.

α2A-AR and TRPM8 are linked via AC and cAMP pathway. A, quantification of the effects of HEK-293_M8 cells pretreatment with AC inhibitor, SQ22536 (200 μm, open column), AC activator, forskolin (10 μm, light gray column), cell membrane-permeable cAMP analog, 8Br-cAMP (1 mm, gray column), or a combination of db-cAMP (1 mm) and phosphodiesterase inhibitor, IBMX (100 μm, dark gray column) on menthol-activated I_TRPM8 (mean ± S.E., n = 6–22 for each condition). B, quantification of the inhibitory effect of clonidine on the density of cold-, icilin-, and menthol-activated I_TRPM8 in HEK-293_M8-α2A-AR cells pretreated (gray columns) and not pretreated (open columns) with forskolin (mean ± S.E., n = 6–10 for each condition). C, averaged time courses of I_TRPM8 (measured as current density at +100 mV) in response to TRPM8-activating stimuli (shown by horizontal bars) in the control HEK-293_M8 cells (black circles) and HEK-293_M8 cells pretreated with β-adrenoreceptor agonist, isoproterenol (1 μm, open circles) (mean ± S.E., n = 6 for each condition). D, same as in C, but for HEK-293_M8-α2A-AR cells under control conditions (black circles) and following treatment with clonidine (open circles) and clonidine plus isoproterenol (black triangles) (mean ± S.E., n = 6 for each condition). E, quantification of the effects of clonidine alone (open columns) and in combination with isoproterenol (gray columns) on the density of cold-, icilin-, and menthol-activated I_TRPM8 (at + 100 mV) in HEK-293_M8-α2A-AR (mean ± S.E., n = 6–10 for each condition). On all graphs (*) and (**) denote statistically significant differences to control with p < 0.05 and p < 0.02, respectively.

FIGURE 4.

Functional link between α2A-AR and TRPM8 is mediated via PKA-dependent phosphorylation. A and B, quantification of the effects of HEK-293_M8 cells pretreatment with PKA inhibitors, KT5720 (1 μm, A) or H-89 (10 μm, B), on the density of cold-, icilin-, and menthol-activated I_TRPM8 in HEK-293_M8 cells (mean ± S.E., n = 5–8 for each condition). C and D, same as in A and B, but for the activators of the serine/threonine protein phosphatase 2A, C2 (10 μm, C) and C6 (10 μm, D) ceramide (mean ± S.E., n = 8 for each condition). On all graphs (*), (**), and (***) denote statistically significant differences to control with p < 0.05, p < 0.02, and p < 0.01, respectively.

FIGURE 5.

Effects of the mutation of TRPM8 PKA phosphorylation sites on the channel activity. A, schematic representation of TRPM8 protein sequence and its putative PKA phosphorylation sites. B, histogram summarizing icilin-activated I_TRPM8 (measured as current density at +100 mV) in HEK-293 cells transfected with TRPM8 (TRPM8) or the mutants S9A, T17A, T32A, S121A, and S367A (mean ± S.E., n = 10–15 for each condition). C, histogram summarizing densities of cold-, icilin-, and menthol-activated I_TRPM8 in HEK-293 cells transfected with TRPM8 (TRPM8) or mutants S9D and T17D in the presence or absence of clonidine (mean ± S.E., n = 5–8 for each condition). On all graphs (*) and (**) denote statistically significant differences to control with p < 0.05 and p < 0.02, respectively.

FIGURE 6.

Functional link between α2A-AR and TRPM8 in native rat DRG neurons. A, representative tracings of the baseline current in DRG neurons and currents in the presence of menthol (100 μm) before and after exposure to clonidine (30 μm) at room temperature. B, averaged time courses of menthol-activated I_TRPM8 and effect on it of clonidine in 5 clonidine-responsive neurons; current was measured at +100 mV and normalized to the maximal value for each neuron (mean ± S.E., n = 5). C, averaged I-V relationships of menthol-activated I_TRPM8 before (black circles) and after (open circles) exposure to clonidine (mean ± S.E., n = 5); inset shows quantification of the effects of clonidine on the density of menthol-activated I_TRPM8 at +100 mV. (*) denotes statistically significant differences with p < 0.05.

FIGURE 7.

Schematic depiction of TRPM8 regulation by α2A- and β-adrenoreceptors. α2A-AR stimulation by clonidine initiates a sequence of intracellular events as follows: Gα_i protein activation following clonidine fixation on α2A-AR leads to AC inhibition and decrease of cAMP production. By consequence, PKA transition from an inactive (i-PKA) to an active (PKA) form is reduced. In parallel, the intracellular serine/threonine protein phosphatase 2A (PP2A) dephosphorylates TRPM8 Ser-9 and Thr-17 inhibiting the channel activity. This inhibitory pathway can be abolished by stimulation of β-AR with isoproterenol, because it would lead to a stimulation of the Gα_s protein coupled to this receptor and to an up-regulation of the adenylate cyclase activity. The subsequent increase in cAMP concentration would potentiate PKA phosphorylation, which will fully maintain the functional activity of TRPM8.