Binding of DL-[³H]-alpha-Amino-3-Hydroxy-5-Methyl-Isoxazole-4-Propionic Acid (AMPA) to Rat Cortex Membranes Reveals Two Sites or Affinity States

Abstract

A method for measuring [³H]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [³H]-AMPA. Non linear curve fitting of [³H]-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 [³H]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [³H]-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 μM); Ibotenic acid and glycine were very weak inhibitors (74 and 92 μM, respectively). CPP, GAMS and L-Aspartic acid showed IC₅₀-values of over 400 μM and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.