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Effects of c‐Src Tyrosine Kinase on Ethanol Sensitivity of Recombinant NMDA Receptors Expressed in HEK 293 Cells
Author(s) -
Anders Douglas L.,
Blevins Tana,
Sutton Greg,
Chandler L. Judson,
Woodward John J.
Publication year - 1999
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/j.1530-0277.1999.tb04122.x
Subject(s) - proto oncogene tyrosine protein kinase src , hek 293 cells , recombinant dna , receptor , tyrosine kinase , chemistry , receptor tyrosine kinase , tropomyosin receptor kinase c , ethanol , microbiology and biotechnology , biochemistry , biology , gene , platelet derived growth factor receptor , growth factor
N ‐Methyl‐D‐aspartate (NMDA) receptors are ligand‐gated ion channels that are important mediators of the actions of the excitatory amino acid neurotransmitter glutamate. Previous studies have shown that ethanol inhibits the function of both wild‐type receptors found in neurons and recombinant NMDA receptors expressed in heterologous cells, such as oocytes and transfected mammalian cells. Although some studies have reported that certain subunit combinations display an enhanced sensitivity to ethanol, this effect is not observed in all experimental systems. This discrepancy may be due to varying levels of endogenous modulators, such as kinases, between different cell preparations. In this study, we investigated the effects of tyrosine phosphorylation on the ethanol sensitivity of NMDA receptor function using a recombinant cell system where levels of both NMDA subunits and protein kinases can be more carefully controlled. Human embryonic kidney (HEK 293) cells were transfected with different NMDA receptor subunits and a c‐Src‐green fluorescent protein (GFP) fusion protein that could be directly visualized in living cells. Agonist‐stimulated calcium flux was measured in single cells using fura‐2 video imaging. As expected, cells transfected with the NR1/NR2B subunits were more sensitive to inhibition by the NR2 selective antagonist ifenprodil than those transfected with NR1/NR2A or NRl/NR2A/NR2B subunits. All receptor combinations were inhibited by ethanol (25 and 100 mM), with the NRl/NR2B combination being slightly more sensitive than NRl/NR2A or NRl/NR2A/NR2B. Control and NMDA‐receptor transfected HEK 293 cells displayed a low degree of tyrosine phosphorylation as measured by immunofluorescence and Western immunoblotting using an anti‐phosphotyrosine antibody. Phosphorylation 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 μM ifenprodil, was not significantly altered by co‐transfection with c‐Src‐GFP. These results indicate that, although NMDA receptors can be a target of c‐Src tyrosine kinase, tyrosine phosphorylation by this enzyme does not modulate the inhibitory effects of ethanol on NMDA‐activated currents.