Characterization of a new AMPA receptor radioligand, [3H]2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid.

(RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), which is a potent and selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, has previously been shown to desensitize AMPA receptors to a much lower degree than AMPA itself. We now report the synthesis of [3H]ACPA (32.5 Ci/mmol), the neurochemical and pharmacological characterization of [3H]ACPA binding, and a comparison of the distribution of [3H]ACPA, [3H]AMPA, and 3H-5-fluorowillardiine binding sites in rat brain. Under equilibrium conditions, [3H]ACPA was shown to bind to a single population of receptor sites on rat brain membranes. [3H]ACPA was shown to bind with single and similar affinities (15-45 nM) to cloned AMPA receptor subunits (GluR1-4), expressed in insect cells, whereas a K(D) value of 330 nM was determined for the binding of [3H]ACPA to cloned kainic acid preferring GluR5 subunits. Whereas Bmax and K(D) values for [3H]ACPA binding, determined using filtration techniques, were different from such obtained in centrifugation assays, Bmax and K(D) values as well as association and dissociation constants were not significantly affected by the addition of the chaotropic agent KSCN. K(D) values, determined under equilibrium conditions, were, however, markedly different from K(D) values derived from kinetic data. Furthermore, the results of analyses of these kinetic data were consistent with the existence of two different populations of [3H]ACPA binding sites. The pharmacology of [3H]ACPA binding sites was characterized using a series of AMPA receptor agonists and antagonists. Whereas addition of KSCN had little effect on the affinities of AMPA receptor agonists for [3H]ACPA binding, this chaotropic agent reduced the affinities of AMPA receptor antagonists structurally related to AMPA. Based on these and previously reported data, the AMPA receptor agonists, ACPA, AMPA and (S)-5-fluorowillardiine, seem to bind to and activate AMPA receptors in a nonidentical fashion, and these three agonists together may be useful tools for studies of AMPA receptor mechanisms.