G_αq Sensitizes TRPM8 to Inhibition by PI(4,5)P₂ Depletion upon Receptor Activation

The cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) channel is important for both physiological temperature detection and cold allodynia. Activation of G-protein-coupled receptors (GPCRs) by proinflammatory mediators inhibits these channels. It was proposed that this inhibition proceeds via direct binding of G_αq to the channel. TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂ or PIP₂] for activity. However, it was claimed that a decrease in cellular levels of this lipid upon receptor activation does not contribute to channel inhibition. Here, we show that supplementing the whole-cell patch pipette with PI(4,5)P₂ reduced inhibition of TRPM8 by activation of G_αq-coupled receptors in mouse dorsal root ganglion (DRG) neurons isolated from both sexes. Stimulating the same receptors activated phospholipase C (PLC) and decreased plasma membrane PI(4,5)P₂ levels in these neurons. PI(4,5)P₂ also reduced inhibition of TRPM8 by activation of heterologously expressed muscarinic M1 receptors. Coexpression of a constitutively active G_αq protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein, decreasing PI(4,5)P₂ levels using a voltage-sensitive 5'-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that, upon GPCR activation, G_αq binding reduces the apparent affinity of TRPM8 for PI(4,5)P₂ and thus sensitizes the channel to inhibition induced by decreasing PI(4,5)P₂ levels.SIGNIFICANCE STATEMENT Increased sensitivity to heat in inflammation is partially mediated by inhibition of the cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) ion channels. Most inflammatory mediators act via G-protein-coupled receptors that activate the phospholipase C pathway, leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. How receptor activation by inflammatory mediators leads to TRPM8 inhibition is not well understood. Here, we propose that direct binding of G_αq both reduces TRPM8 activity and sensitizes the channel to inhibition by decreased levels of its cofactor, PI(4,5)P₂ Our data demonstrate the convergence of two downstream effectors of receptor activation, G_αq and PI(4,5)P₂ hydrolysis, in the regulation of TRPM8.