Labeling of NMDA receptor channels by [3H]MK-801 in brain synaptic membranes treated with Triton X-100.

Binding activity of 3H-5-methyl-10, 11-dihydro-5H-dibenzo [a,d] cyclohepten-5, 10-imine maleate (MK-801) was examined by using rat brain synaptic membranes treated with Triton X-100. This compound is known as a non-competitive antagonist for one subclass of the central excitatory amino acid receptors, N-methyl-D-aspartic acid (NMDA)-sensitive receptors. Triton treatment completely abolished the temperature-dependent portion of the binding activity, with a concomitant reduction of membranous protein content. Addition of L-glutamic acid (Glu), however, markedly potentiated the activity at concentrations higher than 10 nM in a temperature-dependent manner. Similarly significant potentiation was induced by structurally related amino acids as well as agonists for the NMDA-sensitive receptors, but not by agonists for the other subclasses. The rank order of this stimulatory potency was well consistent with that of the displacing activity of these Glu analogues on NMDA-sensitive [3H]Glu binding. Competitive NMDA antagonists, such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid and D-2-amino-5-phosphonovaleric acid, were all effective in preventing the potentiation of [3H]MK-801 binding activity by L-Glu. The latter Glu-dependent activity was additionally enhanced by glycine and its analogues at concentrations above 10 nM in a temperature- and Glu-dependent fashion. Additional enhancement by glycine was also antagonized by competitive NMDA antagonists, but not by a classical glycine antagonist strychnine. These results suggest that Triton X-100-treated membranes are suitable for the study of the interaction of the non-competitive antagonists with NMDA receptor channels, and may be superior to non-detergent-treated membranes in terms of freedom from the confounding effects of endogenous amino acids.