FURTHER EVIDENCE FOR MULTIPLE FORMS OF AN N-METHYL-D-ASPARTATE RECOGNITION DOMAIN IN RAT-BRAIN USING MEMBRANE-BINDING TECHNIQUES

Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsulfonic acid, invariably resulted in marked inhibition of the binding Of -(E)-2-amino-4-[H-3]propyl-5-phosphono-3 -pentenoic acid ([H-3]CGP 39653), a competitive antagonist at an N-met hyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and tre ated with Triton X-1 00, but did not significantly affect the binding of L-[H-3]glutamic acid ([H-3]Glu), an endogenous agonist. The pretrea tment was effective in reducing the binding of [H-3]CGP 39653 at equil ibrium, without altering the initial association rate, and decreased t he affinity for the ligand. Pretreatment with sulfhydryl-reactive agen ts also enhanced the potencies of NMDA agonists to displace [H-3]CGP 3 9653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [H -3]Glu binding. The binding of both [H-3]CGP 39653 and [H-3]Glu was si milarly sensitive to pretreatment with four different proteases in Tri ton-treated membranes, whereas pretreatment with phospholipase A, or C markedly inhibited [H-3]CGP 39653 binding without altering [H-3]Glu b inding. Moreover, both phospholipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [H-3]CGP 396 53 and [H-3]Glu, but also caused diminution of those of NMDA antagonis ts. These results suggest that both sulfhydryl-reactive agents and pho spholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [H-3]CGP 39653, with concomitant facilitation of that in an agonist-preferring form by [H-3]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.