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Activation and Inhibition of Neuronal G Protein-Gated Inwardly Rectifying K⁺ Channels by P2Y Nucleotide Receptors

Department of Pharmacology, University College London, Gower Street, London, United Kingdom (A.K.F., J.M.F.-F., S.J.M., D.A.B.); and Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom (J.S., E.A.B.)

Neuronal signaling by G protein-coupled P2Y nucleotide receptors is not well characterized. We studied here the coupling of different molecularly defined P2Y receptors to neuronal G protein-gated inward rectifier K⁺ (GIRK) channels. Individual P2Y receptors were coexpressed with GIRK1+GIRK2 (Kir3.1 + 3.2) channels by intranuclear plasmid injections into cultured rat sympathetic neurons. Currents were recorded using perforated-patch or whole-cell (disrupted patch) techniques, with similar results. P2Y₁ receptor stimulation with 2-methylthio ADP (2-MeSADP) induced activation of GIRK current (I_GIRK) followed by inhibition. In contrast, stimulation of endogenous ₂-adrenoceptors by norepinephrine produced stable activation without inhibition. P2Y₁-mediated inhibition was also seen when 2-MeSADP was applied after I_GIRK preactivation by norepinephrine or by expression of G₁₂ subunits. In contrast, stimulation of P2Y₄ receptors with UTP or P2Y₆ receptors with UDP produced very little I_GIRK activation but significantly inhibited preactivated currents. Current activation was prevented by pertussis toxin (PTX) or after coexpression of the -scavenger transducin-G.I_GIRK inhibition by all three nucleotide receptors was insensitive to PTX and was significantly reduced after coexpression of RGS2 protein, known to inhibit G_q signaling. Inhibition was not affected 1) after coexpression of RGS11, which interferes with G_q action; 2) after coexpression of phospholipase C (PLC) -Pleckstrin homology domain, which sequesters the membrane phospholipid phosphatidylinositol 4,5-bisphosphate; (3) after buffering intracellular Ca²⁺ with 1,2-bis(2-aminiphenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM); and (4) after pretreatment with the protein kinase C inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF 109203X). We conclude that activation of I_GIRK by P2Y receptors is mediated by G_i/o, whereas I_GIRK inhibition is mediated by G_q. These effects may provide a mechanism for P2Y-modulation of neuronal excitability.

Address correspondence to: Dr. Alexander K. Filippov, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom. E-mail: a.filippov{at}ucl.ac.uk

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