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.
Highlights
Despite being proven as the principal detector of environmental cold [7,8,9], TRPM8 expression is by far not limited to the subset of cold-sensitive dorsal root ganglion and trigeminal sensory neurons in which it functions as cold-activated receptor channel
Pretreatment of HEKTRPM8/M3 cells with 10 M oxotremorine methiodide (Oxo-M) for 10 –20 min resulted in an ϳ3-fold decrease of ITRPM8 density activated by menthol (500 M), which could be prevented by atropine (1 M, Fig. 1B), suggesting that it is a direct consequence of muscarinic receptor stimulation
That stimulation of M3 muscarinic receptors with the nonspecific agonist Oxo-M causes suppression of TRPM8 via a signaling pathway that involves Gq- and Ca2ϩ-independent stimulation of cytosolic phospholipase A2 (cPLA2) and the generation of arachidonic acid (AA), which in turn acts as a direct channel inhibitor (Fig. 4, panel 5)
Summary
Despite being proven as the principal detector of environmental cold [7,8,9], TRPM8 expression is by far not limited to the subset of cold-sensitive dorsal root ganglion and trigeminal sensory neurons in which it functions as cold-activated receptor channel. Consistent with its quite broad expression, TRPM8 function appears to be regulated by cooling temperatures and exogenous chemical imitators of cold, and by a number of second messengers, which are generated during activation of surface receptor-coupled signaling pathways.
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