Characterization of the Binding of [3H]NS 257, a Novel Competitive AMPA Receptor Antagonist, to Rat Brain Membranes and Brain Sections

Abstract: The binding of ³H]NS 257 {1,2,3,6,7,8-hexahydro-3-(hydroxyimino)- N,N -³H]dimethyl-7-methyl-2-oxobenzo2,1- b :3,4- c '] dipyrrole-5-sulfonamide} to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific ³H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 m M . In the presence of thiocyanate ³H]NS 257 bound to a single population of binding sites with an affinity of 225 ± 8 n M and a binding site density of 0.61 ± 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by ³H]NS 257 for both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and l -glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > l -glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of ³H]NS 257 binding sites in rat brain was similar to that of ³H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.