Dl. Anders et al., Effects of c-Src tyrosine kinase on ethanol sensitivity of recombinant NMDA receptors expressed in HEK 293 cells, ALC CLIN EX, 23(2), 1999, pp. 357-362
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.