N-Methyl-D-aspartate (NMDA) receptors (NRs) are ionotropic receptors activated by glutamate and the coagonist glycine. Ethanol inhibits NMDA receptor function, although its site of action is undefined. We hypothesized that ethanol acts at specific amino acids contained within the transmembrane (TM) domains of the receptor. In this study, NR1 and NR2A subunits were altered by mutagenesis and tested for sensitivity to ethanol. Three NR1 mutants (W636A, F817A, and L819A) and one NR2A mutant (F637A) failed to generate functional receptors. Pre-TM1 (I546A, L551A, F554A, and F558A), TM1 (W563A), and TM2 (W611A) NR1 mutations did not affect ethanol sensitivity of heteromeric receptors. In contrast, altering a TM3 phenylalanine to alanine (F639A) reduced the ethanol inhibition of NMDA receptors expressed in oocytes and human embryonic kidney 293 cells. Mutation of the nearby methionine (M641) to alanine did not affect ethanol sensitivity, whereas changing Phe 639 to tryptophan slightly enhanced ethanol inhibition. NR1(F639A) did not alter the agonist potency of glutamate but did produce a leftward shift in the glycine concentration response for receptors containing NR2A and NR2B subunits. NR1(F639A) also reduced the potency of the competitive glycine antagonist 5,7-dichlorokynurenic acid and increased the efficacy of the glycine partial agonist 3-amino-1-hydroxy-2-pyrrolidinone ((؉)-HA-966). These results suggest that ethanol may interact with amino acids contained in the TM3 domain of NMDA subunits that are involved in transducing agonist binding to channel opening. N-Methyl-D-aspartate (NMDA)1 receptors are calcium-permeable ion channels expressed by neurons and require both glutamate and glycine for activation. Combinations of NMDA receptor 1 (NR1) and NR2 subunits yield receptors with different biophysical and pharmacological properties such as differences in desensitization and sensitivity to agonists and antagonists. NMDA receptors play an important role in neuronal development and are required for some forms of synaptic plasticity such as associative long-term potentiation that may underlie some forms of learning and memory (1). NMDA receptors are also involved in the excitotoxic effects of glutamate that accompany traumatic brain injury and stroke-induced ischemia.Ethanol inhibits native NMDA receptor function in vitro and in vivo (2-7). Chronic exposure of neurons to ethanol results in up-regulation of NMDA receptor function and enhanced glutamate-mediated excitotoxicity (8 -10). NMDA antagonists block the seizures associated with ethanol withdrawal (11, 12), and human alcoholics report ethanol-like subjective effects after administration of ketamine, a nondissociative anesthetic that inhibits NMDA channel function (13). Despite the wealth of knowledge indicating that the NMDA receptor is an important target for ethanol in the brain, there is no consensus as to how ethanol inhibits receptor function. Ethanol behaves as a noncompetitive and voltage-independent antagonist of the receptor, and attempts to correlat...

Previous studies in this laboratory have shown that the ethanol inhibition of recombinant NMDA receptors expressed in Xenopus oocytes is subunit‐dependent, with the NR1/2A receptor being more sensitive than NR1/2C receptors. The ethanol sensitivity of NR1/2A receptors is reduced by substitution of the wild‐type NR1‐1a (NR1011) subunit with the calcium‐impermeable NR1 (N616R) subunit. In the present study, the ethanol inhibition of NMDA receptors was determined under different conditions to examine the role that calcium plays in determining the ethanol sensitivity of recombinant NMDA receptors. The ethanol sensitivity of NR1/2B or NR1/2C receptors was not affected by alterations in extracellular calcium levels or by coexpression with calcium‐impermeable NR1 mutants. In contrast, the inhibition of NR1/2A receptors by 100 mM ethanol was reduced in divalent‐free recording medium and was significantly increased when 10 mM calcium was used as the only charge carrier. The increase in the ethanol sensitivity of NR1/2A receptors under high‐calcium conditions was prevented by preinjection of oocytes with the calcium chelator 1,2‐bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) but not by inhibitors of calmodulin or protein kinase C. Ethanol did not alter the channel blocking activity of divalent cations on NMDA‐induced currents. The enhanced ethanol sensitivity of NR1/2A receptors in 10 mM calcium persisted when the NR1 subunit was replaced by the alternative splice variant NR1‐4a (NR1000), which lacks the C1 and C2 cassettes. However, expression of a mutant NR1 subunit that lacked the C0, C1, and C2 domains abolished the calcium‐dependent enhancement of ethanol's inhibition of NR1/2A receptors. Finally, the ethanol sensitivity of wild‐type NR1/2A receptors measured in transfected HEK 293 cells by whole cell patch‐clamp electrophysiology was significantly reduced by expression of the C‐terminal truncated NR1 subunit. These results demonstrate that the ethanol sensitivity of certain NMDA receptors is modulated by an intracellular, calcium‐dependent process that requires the C0 domain of the NR1 subunit.

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