BINDING OF DL-[H-3]-ALPHA-AMINO-3-HYDROXY-5-METHYL-ISOXAZOLE-4-PROPIONIC ACID (AMPA) TO RAT CORTEX MEMBRANES REVEALS 2 SITES OR AFFINITY STATES

A method for measuring [H-3]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [H-3]-AMPA. Non linear curve fitting of [H-3]-AMPA saturation isotherms suggested the presence of two binding sites: the high affinity site showed a pKd of 8.26 +/- 0.07 (Kd = 5.49 nM) and a Bmax of 0.19 +/- 0.03 pmol/mg protein, whereas the low affinity site indicated a pKd of 7.28 +/- 0.05 (Kd = 52 nM) and a Bmax of 1.30 +/- 0.23 pmol/mg protein. The pharmacological profile of [H-3]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [H-3]-AMPA binding (IC50 = 9.7 nM), followed by AMPA (19 nM), CNQX, DNQX and L-Glutamate (272-373 nM). Kainate was a moderate displacer (6.2-mu-M); Ibotenic acid and glycine were very weak inhibitors (74 and 92-mu-M, respectively). CPP, GAMS and L-Aspartic acid showed IC50-values of over 400-mu-M and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.