High-affinity [H-3] kainic acid binding to brain membranes: a re-evaluation of ligand potency and selectivity

[H-3]Kainic acid ([H-3]KA) is a widely used tool for studying the KA class of excitatory amino acid receptors. [H-3]KA of significantly higher specifi c activity has become available permitting use of radioligand concentration s below the dissociation constant (K-D) of the high-affinity binding site. We employed low radioligand (0.05-0.2 nM) and receptor concentrations (0.01 nM) to gain new insights into the binding characteristics of the high-affi nity KA binding site in a standard preparation of lyzed synaptosomal membra nes from the: cerebral cortex of male Sprague-Dawley rats. Under these cond itions, KA binds to a single class of high-affinity sites with a K-D of 1.0 +/- 0.3 nM. The potencies of competing agents are considerably higher than published reports. Specifically, domoic acid, glutamate, and glutamine exh ibit IC50 values for displacing [H-3]KA of 0.37 +/- 0.02, 94 +/- 13, and 15 00 +/- 500 nM, respectively. Domoate (1 mu M) was tested against a panel of 32 central nervous system binding sites and found to be inactive at each, indicating this toxin displays considerable selectivity. This study illustr ates the remarkable potency of domoic acid and underlines the importance of performing radioligand binding studies at concentrations of constituents t hat permit characterization of high-affinity interactions. (C) 2000 Elsevie r Science Inc. All rights reserved.