Effects of c-Src tyrosine kinase on ethanol sensitivity of recombinant NMDA receptors expressed in HEK 293 cells

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that ar e important mediators of the actions of the excitatory amino acid neurotran smitter glutamate. Previous studies have shown that ethanol inhibits the fu nction of both wild-type receptors found in neurons and recombinant NMDA re ceptors expressed in heterologous cells, such as oocytes and transfected ma mmalian cells. Although some studies have reported that certain subunit com binations display an enhanced sensitivity to ethanol, this effect is not ob served in all experimental systems. This discrepancy may be due to varying levels of endogenous modulators, such as kinases, between different cell pr eparations. In this study, we investigated the effects of tyrosine phosphor ylation on the ethanol sensitivity of NMDA receptor function using a recomb inant cell system where levels of both NMDA subunits and protein kinases ca n be more carefully controlled. Human embryonic kidney (HEK 293) cells were transfected with different NMDA receptor subunits and a c-Src-green fluore scent protein (GFP) fusion protein that could be directly visualized in liv ing cells. Agonist-stimulated calcium flux was measured in single cells usi ng fura-2 video imaging, As expected, cells transfected with the NR1/NR2B s ubunits were more sensitive to inhibition by the NR2 selective antagonist i fenprodil than those transfected with NR1/ NR2A or NR1/NR2A/NR2B subunits. All receptor combinations were inhibited by ethanol (25 and 100 mM), with t he NR1/NR2B combination being slightly more sensitive than NR1/NR2A or NR1/ NR2A/ NR2B, Control and NMDA-receptor transfected HEK 293 cells displayed a low degree of tyrosine phosphorylation as measured by immunofluorescence a nd Western immunoblotting using an antiphosphotyrosine antibody. Phosphoryl ation was markedly enhanced in cells transfected with the c-Src-GFP fusion protein. The sensitivity of NMDA receptors to either 25 or 100 mM ethanol, or 10 mu M ifenprodil, was not significantly altered by co-transfection wit h c-Src-GFP, These results indicate that, although NMDA receptors can be a target of c-Src tyrosine kinase, tyrosine phosphorylation by this enzyme do es not modulate the inhibitory effects of ethanol on NMDA-activated current s.