Further evidence for multiple forms of an N-methyl-D-aspartate recognition domain in rat brain using membrane binding...

Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsulfonic acid, invariably resulted in marked inhibition of the binding of DL-(E)-2-amino-4-[3H]propyl-5-phosphono-3-pentenoic acid ([3H]CGP 39653), a competitive antagonist at an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[3H]-glutamic acid ([3H]Glu), an endogenous agonist. The pretreatment was effective in reducing the binding of [3H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [3H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [3H]Glu binding. The binding of both [3H]CGP 39653 and [3H]Glu was similarly sensitive to pretreatment with four different proteases in Triton-treated membranes, whereas pretreatment with phospholipase A2 or C markedly inhibited [3H]CGP 39653 binding without altering [3H]Glu binding. Moreover, both phospholipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [3H]CGP 39653 and [3H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl-reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [3H]CGP 39653, with concomitant facilitation of that in an antagonist-preferring form by [3H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.