Ethanol inhibition of recombinant NR1/2A receptors: effects of heavy metal chelators and a zinc-insensitive NR2A mutant

Abstract

N-methyl- d-aspartate (NMDA) receptors are a subtype of ionotropic glutamate receptors that are highly expressed in brain neurons. These receptors are calcium permeable and regulate various forms of plasticity, including long-term potentiation of glutamate synapses. Acutely, ethanol inhibits the function of NMDA receptors, and chronic exposure of neurons to ethanol is associated with enhanced NMDA receptor function during withdrawal. Results from previous studies with recombinant NMDA receptors have demonstrated that subunit composition influences the ethanol sensitivity of NMDA receptors, with NR2A-containing receptors often showing greater inhibition by ethanol than shown by those containing other NR2 subunits. NR2A-containing NMDA receptors are particularly sensitive to other modulators of channel function, including heavy metals such as zinc. Although zinc inhibits NMDA receptors at micromolar concentrations through a voltage-dependent interaction with a channel site, NR2A-containing receptors are inhibited by low nanomolar concentrations of zinc in a voltage-independent manner. In this study, we examined the effects of ethanol on NR1/NR2A receptors expressed in human embryonic kidney 293 (HEK 293) cells recorded under conditions in which the effects of zinc are minimized. Under control conditions, ethanol (100 mM) inhibited peak and steady-state currents of NR1/2A receptors by 35%–40%. Inclusion of the heavy metal chelator ethylenediaminetetraacetic acid [(EDTA); 10 μM] or other chelating compounds in the experimental solution augmented NMDA-stimulated currents, as previously described. In the presence of EDTA (10 μM), the inhibition of NR1/2A currents by 100 mM ethanol was reduced to approximately 28%. As expected, currents recorded from cells expressing the NR1 subunit and a zinc-insensitive NR2A mutant (H128S) were not augmented by zinc chelators. In these cells, ethanol (100 mM) inhibited NMDA receptor currents by approximately 28%, and this inhibition was unaffected by EDTA. These results support the suggestion that low levels of zinc present in experimental solutions may affect the apparent ethanol sensitivity of NMDA receptors containing the NR2A subunit.