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

• Published in: Molecular pharmacology 2004-09, Vol.66 (3), p.468-477
• Main Authors: Filippov, Alexander K, Fernandez-Fernandez, Jose M, Marsh, Stephen J, Simon, Joseph, Barnard, Eric A, Brown, David A
• Format: Article
• Language: English
• ISSN: 0026-895X

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 super(+) (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 sub(1) receptor stimulation with 2- methylthio ADP (2-MeSADP) induced activation of GIRK current (I sub(GIRK)) followed by inhibition. In contrast, stimulation of endogenous alpha sub(2)- adrenoceptors by norepinephrine produced stable activation without inhibition. P2Y sub(1)-mediated inhibition was also seen when 2-MeSADP was applied after I sub(GIRK) preactivation by norepinephrine or by expression of G beta sub(1)gamma sub(2) subunits. In contrast, stimulation of P2Y sub(4) receptors with UTP or P2Y sub(6) receptors with UDP produced very little I sub(GIRK) activation but significantly inhibited preactivated currents. Current activation was prevented by pertussis toxin (PTX) or after coexpression of the beta gamma-scavenger transducin- Galpha.I sub(GIRK) inhibition by all three nucleotide receptors was insensitive to PTX and was significantly reduced after coexpression of RGS2 protein, known to inhibit G sub(q)alpha signaling. Inhibition was not affected 1) after coexpression of RGS11, which interferes with G sub(q) beta gamma 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 super(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 sub(GIRK) by P2Y receptors is mediated by G sub(i/o) beta gamma, whereas I sub(GIRK) inhibition is mediated by G sub(q)alpha. These effects may provide a mechanism for P2Y-modulation of neuronal excitability.