Effects of ethanol on phosphorylation site mutants of recombinant N-methyl-D-aspartate receptors.

N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by the neurotransmitter glutamate. These channels are highly expressed by brain neurons and are critically involved in excitatory synaptic transmission. Results from previous studies show that both native and recombinant NMDA receptors are inhibited by ethanol at concentrations associated with signs of behavioral impairment and intoxication. Given the important role that NMDA receptors play in synaptic transmission and brain function, it is important to understand the factors that regulate the ethanol inhibition of these receptors. One dynamic mechanism for regulating ethanol action may be via phosphorylation of NMDA subunits by serine-threonine and tyrosine kinases. Both NR1 and NR2 subunits contain multiple sites of phosphorylation; and in the NR1 subunit, most of these are contained within the C1 domain, a carboxy-terminal cassette that is subject to alternative splicing. Although results from our previous studies suggest that single phosphorylation sites do not greatly affect ethanol sensitivity of NMDA receptors, it is likely that in vivo, these subunits are phosphorylated at multiple sites by different kinases. In the present study, we constructed a series of NMDA receptor mutants at serine (S) or threonine (T) residues proposed to be sites of phosphorylation by protein kinase A and various isoforms of protein kinase C. Ethanol (100mM) inhibited currents from wild-type NR1/2A and NR1/2B receptors expressed in human embryonic kidney 293 cells by approximately 25 and 30%, respectively. This inhibition was not different in single-site mutants expressing alanine (A) or aspartate/glutamate (D/E) at positions T879, S896, or T900. The mutant NR1(S890D) showed greater ethanol inhibition than NR1(890A) containing receptors, although this was only observed when it was combined with the NR2A subunit. Ethanol inhibition was not altered by aspartate substitution at four serines (positions 889, 890, 896, and 897) or when T879D was added to the four serine-substituted mutant. Ethanol inhibition was increased when T900E was added to the five serine-/threonine-substituted mutants, but again this was selective for NR2A containing receptors. Together with previously published data, these findings suggest that modification of putative phosphorylation sites could contribute to the overall acute ethanol sensitivity of recombinant NMDA receptors. Supported by R37AA009986.