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Filippov AK, Fernández-Fernández JM, Marsh SJ, Simon J, Barnard EA, Brown DA

Activation and inhibition of neuronal G protein-gated inwardly rectifying K(+) channels by P2Y nucleotide receptors.

Mol. Pharmacol.. 2004 Sep;66(3):468-77, PMID: 15322238

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. P2Y(1) receptor stimulation with 2-methylthio ADP (2-MeSADP) induced activation of GIRK current (I(GIRK)) followed by inhibition. In contrast, stimulation of endogenous alpha(2)-adrenoceptors by norepinephrine produced stable activation without inhibition. P2Y(1)-mediated inhibition was also seen when 2-MeSADP was applied after I(GIRK) preactivation by norepinephrine or by expression of Gbeta(1)gamma(2) subunits. In contrast, stimulation of P2Y(4) receptors with UTP or P2Y(6) 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 betagamma-scavenger transducin-Galpha.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)alpha signaling. Inhibition was not affected 1) after coexpression of RGS11, which interferes with G(q)betagamma action; 2) after coexpression of phospholipase C (PLC) delta-Pleckstrin homology domain, which sequesters the membrane phospholipid phosphatidylinositol 4,5-bisphosphate; (3) after buffering intracellular Ca(2+) 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)betagamma, whereas I(GIRK) inhibition is mediated by G(q)alpha. These effects may provide a mechanism for P2Y-modulation of neuronal excitability.

PHARMACOLOGY & PHARMACY