Differential effects of TM4 tryptophan mutations on inhibition of N-methyl-d-aspartate receptors by ethanol and toluene.

The voluntary use and abuse of alcohol and inhalants is a recognized health problem throughout the world. Previous studies have shown that these agents affect brain function in a variety of ways including direct inhibition of key ion channels that regulate neuronal excitability. Among these, the N-methyl-d-aspartate (NMDA) receptor is particularly important given its key role in glutamatergic synaptic transmission, neuronal plasticity and learning and memory. Previous studies from this laboratory and others have identified key residues within transmembrane (TM) domains of the NMDA receptor that appear to regulate its sensitivity to alcohol and anesthetics. In this study, we extend these findings and examine the role of a TM4 residue in modulating sensitivity of recombinant NMDA receptors to ethanol and toluene. HEK293 cells were transfected with GluN1-1a and either wild-type or tryptophan-substituted GluN2(A-D) subunits and whole-cell currents were recorded using patch-clamp electrophysiology in the absence or presence of ethanol or toluene. Both ethanol (100 mM) and toluene (1 or 3 mM) reversibly inhibited glutamate-activated currents from wild-type NMDARs with GluN2B containing receptors showing heightened sensitivity to either agent. Substitution of tryptophan (W) at positions 825, 826, 823 or 850 in the TM4 domain of GluN2A, GluN2B, GluN2C or GluN2D subunits; respectively, significantly reduced the degree of inhibition by ethanol. In contrast, toluene inhibition of glutamate-activated currents in cells expressing the TM4-W mutants was not different from that of the wild-type controls. These data suggest that despite similarities in their action on NMDARs, ethanol and toluene may act at different sites to reduce ion flux through NMDA receptors.