Ethanol sensitivity of heteromeric NMDA receptors: Effects of subunit assembly, glycine and NMDAR1 Mg 2+-insensitive mutants

Abstract

In the current study, dimeric and trimeric combinations of N-methyl- d-aspartate (NMDA) receptor subunits were expressed in Xenopus oocytes and their sensitivity to ethanol was examined using conventional two electrode voltage clamp methods. In oocytes expressing the NR1/2A subunits, ethanol (25, 50 and 100 mM) inhibited NMDA (100 μM)/glycine (10 μM) induced currents by 21, 31 and 47%; respectively. NMDA-stimulated currents in oocytes expressing NR1/2B currents were inhibited by 13, 25 and 45% while NR1/NR2C currents were inhibited by 6, 11 and 24%. Ethanol inhibition of NMDA-stimulated currents in oocytes injected with NR1/2A/2B or NR1/2A/2C was not significantly different from that observed in NR1/2B or NR1/2C injected oocytes, respectively. With all receptor combinations, ethanol inhibition was rapid, reversible and not altered by pre-incubation. In the absence of ethanol, glycine enhanced NMDA-induced currents with an EC 50 of 1.42 μM for the NR1/NR2A combination and 0.51 μM for the NR1/NR2C combination. Ethanol inhibited NMDA-induced currents at all glycine concentrations tested (1–100-μM) and did not significantly alter the EC 50 value for glycine suggesting that ethanol does not compete for the glycine site on the NMDA receptor. Finally, three NR1 mutants which have been previously shown by others to possess either decreased Mg 2+ sensitivity and Ca 2+ permeability (N616Q and N616R) or reduced current amplitude (F609L) were tested for their ethanol sensitivity when expressed in combination with the NR2A subunit. Substitution of the wild-type NR1 with F609L did not alter the sensitivity of the receptor to ethanol. However NR1(N616Q)/NR2A and NR1 (N616R)/NR2A injected oocytes showed a reduced sensitivity to ethanol suggesting that alterations in the ion permeation characteristics of recombinant NMDA receptors may influence their sensitivity to ethanol.