Acute effects of ethanol on recombinant kainate receptors: lack of role of protein phosphorylation.
Valenzuela CF., Cardoso RA., Lickteig R., Browning MD., Nixon KM.
This study examined the acute actions of ethanol on recombinant rat GluR6 kainate receptors expressed in Xenopus oocytes and HEK 293 cells. Electrophysiological recordings showed that co-application of ethanol with submaximal kainate concentrations resulted in similar inhibition of kainate-gated currents in both expression systems. Manipulation of intracellular phosphorylation pathways by intracellular dialysis with a solution without ATP and GTP did not modify the inhibitory effects of ethanol. Moreover, co-transfection of GluR6 receptor subunits with PKA-alpha catalytic subunit or the calcium/calmodulin-dependent protein kinase II (CamKII) catalytic fragment did not change the sensitivity of the receptor to ethanol. Treatment of Xenopus oocytes with specific inhibitors of PKC, PKA, CamKII, tyrosine kinases, and serine-threonine protein phosphatases did not affect the 100 mM ethanol-induced inhibition of GluR6 receptor-mediated currents. Biochemical experiments with transiently transfected HEK 293 cells confirmed published reports that GluR6 receptors are minimally phosphorylated under basal conditions in these cells and also revealed that acute ethanol did not increase GluR6 phosphorylation. These results suggest that, under our experimental conditions, ethanol inhibits recombinant GluR6 receptor function by a direct effect on the receptor rather than an indirect action via protein phosphorylation.