Complex regulation of the TRPM8 cold receptor channel: role of arachidonic acid release following M3 muscarinic receptor stimulation

Abstract

Cold/menthol-activated TRPM8 (transient receptor potential channel melastatin member 8) is primarily expressed in sensory neurons, where it constitutes the principal receptor of environmental innocuous cold. TRPM8 has been shown to be regulated by multiple influences such as phosphorylation, pH, Ca(2+), and lipid messengers. One such messenger is arachidonic acid (AA), which has been shown to inhibit TRPM8 channel activity. However, the physiological pathways mediating the inhibitory effect of AA on TRPM8 still remain unknown. Here, we demonstrate that TRPM8 is regulated via M3 muscarinic acetylcholine receptor-coupled signaling cascade based on the activation of cytosolic phospholipase A2 (cPLA2) and cPLA2-catalyzed derivation of AA. Stimulation of M3 receptors heterologously co-expressed with TRPM8 in HEK-293 cells by nonselective muscarinic agonist, oxotremorine methiodide (Oxo-M), caused inhibition of TRPM8-mediated membrane current, which could be mimicked by AA and antagonized by pharmacological or siRNA-mediated cPLA2 silencing. Our results demonstrate the intracellular functional link between M3 receptor and TRPM8 channel via cPLA2/AA and suggest a novel physiological mechanism of arachidonate-mediated regulation of TRPM8 channel activity through muscarinic receptors. We also summarize the existing TRPM8 regulations and discuss their physiological and pathological significance.

FIGURE 1.

Agonist-induced stimulation of muscarinic receptors causes inhibition of TRPM8. A, effect of Oxo-M (10 μm) on [Ca²⁺]_i in HEK_TRPM8/M3 cells in Ca²⁺-free EGTA-containing extracellular medium. Incubation with atropine (Atrop.; 1 μm) antagonized responses to Oxo-M, confirming that responses were mediated by activation of muscarinic receptors (mean ± S.E., n = 25–50). B, averaged I-V relationships (mean ± S.E., n = 5) of menthol-activated (500 μm) I_TRPM8 density under control conditions (black symbols), in the presence of Oxo-M (10 μm, open circles) alone and Oxo-M plus atropine (1 μm, gray triangles); the inset shows quantification of the respective I_TRPM8 densities at +100 mV. C, quantification of I_TRPM8 density (at +100 mV) activated by cold (temperature drop from 33 to 20 °C), icilin (10 μm), and menthol (500 μm) in HEK_TRPM8/M3 cells under control conditions (black bars) and in the presence of Oxo-M (10 μm, white bars) (mean ± S.E., n = 6). On all graphs, double asterisks denote statistically significant differences to control with p < 0.02.

FIGURE 2.

Functional link between muscarinic receptors and TRPM8 is mediated via the cPLA2/AA pathway. A, quantification of I_TRPM8 density (at +100 mV) activated by cold, icilin, and menthol in HEK_TRPM8/M3 cells under control conditions (black bars), in the presence of Oxo-M (white bars) and Oxo-M plus PLC inhibitor U73122 (1 μm, gray bars) (mean ± S.E., n = 4–6). B, averaged I-V relationships (mean ± S.E., n = 6) of menthol-activated I_TRPM8 density under control conditions (filled symbols) and in the presence of arachidonic acid (AA, 10 μm, open circles); the inset shows quantification of the respective I_TRPM8 densities at +100 mV. C, quantification of I_TRPM8 density (at +100 mV) activated by icilin and menthol in HEK_TRPM8/M3 cells under control conditions (black bars) and in the presence of Oxo-M during cell dialysis with control intracellular solution (white bars) and the one supplemented with cPLA2 inhibitor AACOCF3 (50 μm, gray bars) (mean ± S.E., n = 4–6). D, quantification of I_TRPM8 density (at +100 mV) activated by cold, icilin, and menthol in the control HEK_TRPM8/M3 cells (i.e. transfected with siLuc) under control conditions (black bars) and following application of Oxo-M (white bars) as well as in HEK_TRPM8/M3 cells transfected with anti-cPLA2 siRNA (sicPLA2) under the action of Oxo-M (gray bars) (mean ± S.E., n = 4–6). For all data presented, I_TRPM8 was activated by temperature drop from 33 to 20 °C (cold), icilin (10 μm), or menthol (500 μm), and Oxo-M was used at 10 μm. On all graphs, single and double asterisks denote statistically significant differences to control with p < 0.05 and p < 0.02, respectively.

FIGURE 3.

Functional link between muscarinic receptors and TRPM8 in PC-3 cells. A, averaged time course of I_TRPM8 (measured as current density at +100 mV) in response to TRPM8-activating stimuli (shown by horizontal lines): temperature drop from 33 to 20 °C (cold), icilin (10 μm), and menthol (500 μm) in PC-3 cells transiently transfected with the human TRPM8 under control conditions (filled circles) or following treatment with Oxo-M (10 μm, open circles). B, quantification of I_TRPM8 density (at +100 mV) activated by cold, icilin, and menthol in PC-3 cells transiently transfected with human TRPM8 under control conditions (black bars) or in the presence of Oxo-M (white bars) (mean ± S.E., n = 5). On all graphs, single and double asterisks denote statistically significant differences to control with p < 0.05 and p < 0.02, respectively.

FIGURE 4.

Schematic of principal signaling pathways involved in plasmalemmal TRPM8 regulation. Each numbered box depicts molecular interconnection related to the specific signaling pathway. Box 1, PIP₂ depletion pathway. PIP₂ is a key lipid co-factors in TRPM8 activation, which depletion as a result of Gq-coupled receptor-mediated stimulation (PDGF receptor depicted) of PLC reduces TRPM8 functionality. Box 2, iPLA2/LPL pathway. The products of iPLA2 catalytic activity, LPLs, act as TRPM8 activators; stimulation of iPLA2 and consequently enhancement of LPL production can be achieved by endoplasmic reticulum (ER) Ca²⁺ store depletion. Box 3, the cAMP/PKA pathway. Decrease of the basal level of cAMP/PKA-dependent phosphorylation of TRPM8 via recruitment of the α2-adrenoreceptor (α2AR)-coupled Gi/AC/cAMP/PKA pathway inhibits channel function; the level of cAMP/PKA-dependent phosphorylation, and consequently, TRPM8 activation can be restored via the Gs/AC/cAMP/PKA pathway coupled to β-adrenoreceptor (βAR). Box 4, PLC/PKC pathway. The PSA can activate TRPM8 via the bradykinin receptor (B2R) signaling pathway involving protein kinase C (PKC) Ca²⁺-dependent activation of PKC may also indirectly contribute to the Ca²⁺-mediated TRPM8 channel desensitization. Box 5, M3 muscarinic receptor-stimulated cPLA2/AA pathway identified in this study. AA derived as a result of nonclassical M3 muscarinic receptor-mediated stimulation of cPLA2 (marked by ?) acts as a TRPM8 channel inhibitor. DAG, diacylglycerol; ISO, isoproterenol; PDGFR, PDGF receptor. Green circles with + and green arrows indicate stimulatory action, red circles with − and red arrows indicate inhibitory action; and a downward red arrow near PIP₂ indicates decreasing of its content.