Further Evidence for Multiple Forms of an N‐Methyl‐d‐Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques

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Abstract
Abstract— Pretreatment with sulfhydryl‐reactive agents, such as N‐ethylmaleimide and p‐chloromercuriphenylsul‐fonic 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 pre‐treatment 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 Tritontreated membranes, whereas pretreatment with phospho‐lipase A2 or C markedly inhibited [3H]CGP 39653 binding without altering [3H]Glu binding. Moreover, both phospho‐lipases 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 agonist‐preferring form by [3H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.

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