Abstract
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 (IGIRK) followed by inhibition. In contrast, stimulation of endogenous α2-adrenoceptors by norepinephrine produced stable activation without inhibition. P2Y1-mediated inhibition was also seen when 2-MeSADP was applied after IGIRK preactivation by norepinephrine or by expression of Gβ1γ2 subunits. In contrast, stimulation of P2Y4 receptors with UTP or P2Y6 receptors with UDP produced very little IGIRK activation but significantly inhibited preactivated currents. Current activation was prevented by pertussis toxin (PTX) or after coexpression of the βγ-scavenger transducin-Gα.IGIRK inhibition by all three nucleotide receptors was insensitive to PTX and was significantly reduced after coexpression of RGS2 protein, known to inhibit Gqα signaling. Inhibition was not affected 1) after coexpression of RGS11, which interferes with Gqβγ action; 2) after coexpression of phospholipase C (PLC) δ-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 IGIRK by P2Y receptors is mediated by Gi/oβγ, whereas IGIRK inhibition is mediated by Gqα. These effects may provide a mechanism for P2Y-modulation of neuronal excitability.
- Received February 27, 2004.
- Accepted June 2, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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