| N-Methyl-D-Aspartate (NMDA) receptors are excitatory ionotropic glutamate receptors composed of at least one NR1 subunit and one or more members of the NR2 (A-D) subunit family. Ethanol inhibits NMDA receptors but the mechanism of action is unknown. We hypothesize that ethanol acts on the NMDA receptor by partitioning into an area defined by specific amino acids. In these studies, specific amino acids in, or near, the transmembrane (TM) domains of the NR1 subunit have been mutated to alanine, co-expressed with wild-type NR2A subunits in Xenopus oocytes, and tested for sensitivity to ethanol. The oocytes were voltage-clamped at -80 mV and challenged with ethanol during activation with NMDA and glycine in a barium-containing buffer. In the pre-TM1 region, NR1 mutants I546A, L551A, F554A, and F558A showed no changes in ethanol sensitivity compared to wild-type NR1/2A receptors. The TM1 mutant W563A and the TM2 mutant W611A were inhibited by ethanol to the same extent as wild-type receptors. The NR1 TM3 mutant F639A significantly decreased ethanol inhibition of the receptor when expressed with NR2A, NR2B, or NR2C subunits. This effect was unlikely to be due to non-specific disruption of receptor structure because alteration of a neighboring amino acid, M641 did not affect the ethanol sensitivity of the receptor. Mutation of F639 to the larger tryptophan did not significantly alter the ethanol sensitivity of these receptors. To further characterize the NR1(F639A)/2A mutant receptor, glutamate concentration-response experiments were performed and a small difference was found between mutant and wild-type receptors. Glycine concentration-response curves were significantly different for NR1(F639A)/2A and NR1(F639A)/2B and their respective wild-type receptors, but no difference was found for NR1(F639A)/2C receptors. NR1(F639A)/2A receptors were activated to a greater extent by the partial-agonist (+)-HA-966 than wild-type receptors, suggesting that this mutant may enhance glycine's ability to open the channel. Taken together, these results suggest that F639 may represent an important site that regulates the overall ethanol sensitivity of NMDA receptors. The work was supported by AA09986 (JJW), K02AA00238 (JJW), and T32-DA07027 (KMR). |
