Dephosphorylation of GluN2B C-terminal tyrosine residues does not contribute to acute ethanol inhibition of recombinant NMDA receptors.
N-methyl-d-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10-600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors.
Department of Neurosciences and Center for Drug and Alcohol Programs, Medical University of South Carolina, Charleston, SC 29425, USA.
Benjamin A
BA
Hughes
C Thetford
CT
Smothers
John J
JJ
Woodward
D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D000431:Ethanol; D014443:Tyrosine