Effects of thiol-reagents on [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding to rat...

The binding of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a ligand for the quisqualate subtype of excitatory amino acid receptors, was measured after chemical modifications of rat brain synaptic membranes. Treatment with oxidizing or thiol-alkylating agents did not modify [3H]AMPA binding, whereas treatment with several sulfhydryl reagents produced marked increases in binding. The involvement of free sulfhydryl groups in the regulation of the properties of [3H]AMPA binding sites was suggested by the specificity of p-chloromercuribenzoic acid (PCMB), its sulfonate analog p-chloromercuriphenyl-sulfonic acid (PCMBS), and HgCl2, plus the reversal of their effects after reduction with dithiothreitol. Pretreatment of synaptic membranes with the oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) or the alkylating agent N-ethylmaleimide did not significantly affect [3H]AMPA binding but markedly reduced the enhancing effect of PCMBS. On the other hand, the increase in [3H]AMPA binding produced by PCMBS was not prevented by treatment with agonists such as quisqualate or L-glutamate and was produced equally well in resealed postsynaptic membranes with both lipophilic or nonlipophilic SH-reagents. Using filtration assays, two types of binding sites could be detected with high and low affinity for [3H]AMPA. Treatment with SH-reagents produced an increase in the Bmax for the high affinity component and a decrease in the Bmax for the low affinity component, accompanied by an increase in its affinity for the ligand. Using centrifugation assays, the same two types of sites could be detected under control conditions but treatment with SH-reagents produced an increase in affinity of the large component that prevented the analytical differentiation of the two sites. Treatment with SH-reagents also increased the binding of [3H] glutamate to the N-methyl-D-aspartate receptors but did not modify the binding of [3H]kainate to the kainate receptors or the strychnine-insensitive [3H]glycine binding. These results suggest that free sulfhydryl groups allosterically modulate the affinity of the quisqualate subtype of excitatory amino acid receptors and also indicate that different types of glutamate receptors might be differentially affected by chemical modification.