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The binding of [(3)H]?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) to synaptic membranes of rat brain was characterized in the absence of detergents and chaotropes. Optimal conditions for binding were a centrifugation assay employing a well washed, frozen-thawed synaptic membrane preparation and a 40 min incubation at 4 degrees C in 50 mM Tris-HC1/100 mM KC1, pH 7.4, buffer. Under these conditions, saturation experiments showed [(3)H]AMPA bound to high and low affinity sites (approx. K(d)s 150 nM and 15 ?M, respectively). Both components were also detectable when binding was terminated by filtration, but B(max) values were 20 times lower than found by centrifugation. Kainic acid (3 ?M) blocked binding of [(3)H]AMPA to the low affinity site, and competition studies at the high affinity site yielded a pharmacological profile consistent with that of a quisqualate receptor. Rank order of potency of putative agonists quisqualate > glutamate > AMPA > 4-methylhomoibotenate ??-N- oxalyl- l -?,?-diaminopropionate > willardine . A number of quinoxalines also inhibited all specific binding of [(3)H]AMPA, but glutamate diethylester was a weak inhibitor. Barbiturates failed to displace binding of [(3)H]AMPA, and had no effects on association and dissociation. Binding conditions were applicable to slide-mounted sections of brain (70-90% specific binding) and the autoradiographic localization of high affinity, quisqualate-sensitive sites labelled by [(3)H]AMPA. The quisqualate receptor can be successfully studied with [(3)H]AMPA in the absence of detergents and chaotropes, but special attention needs to be given to low affinity kainate-sensitive sites. |
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