Mechanism of inhibition of TREK-2 (K2P10.1) by the Gq-coupled M3muscarinic receptor

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Abstract
TREK-2 is a member of the two-pore domain K+channel family and provides part of the background K+current in many types of cells. Neurotransmitters that act on receptors coupled to Gqstrongly inhibit TREK-2 and thus enhance cell excitability. The molecular basis for the inhibition of TREK-2 was studied. In COS-7 cells expressing TREK-2 and M3receptor, acetylcholine (ACh) applied to the bath solution strongly inhibited the whole cell current, and this was markedly reduced in the presence of U-73122, an inhibitor of PLC. The inhibition was also observed in cell-attached patches when ACh was applied to the bath solution. In inside-out patches, direct application of guanosine 5′- O-(3-thiotriphosphate) (10 μM), Ca2+(5 μM), or diacylglycerol (DAG; 10 μM) produced no inhibition of TREK-2 in >75% of patches tested. Phosphatidic acid, a product of DAG kinase, had no effect on TREK-2. Pretreatment of cells with 20 μM wortmannin, an inhibitor of phosphatidylinositol kinases, did not affect the inhibition or the recovery from inhibition of TREK-2, suggesting that phosphatidylinositol 4,5-bisphosphate depletion did not mediate the inhibition. Pretreatment of cells with a protein kinase C inhibitor (bisindolylmaleimide, 10 μM) markedly inhibited ACh-induced inhibition of TREK-2. Mutation of two putative PKC sites (S326A, S359C) abolished inhibition by ACh. Mutation of these amino acids to aspartate to mimic the phosphorylated state resulted in diminished TREK-2 current and no inhibition by ACh. These results suggest that the agonist-induced inhibition of TREK-2 via M3receptor occurs primarily via PKC-mediated phosphorylation.