Binding Sites for l‐[3H]Glutamate on Hippocampal Synaptic Membranes: Three Populations Differentially Affected by Chloride and Calcium Ions

Abstract

The effects of Cl- and Ca2- were studied on the specific binding of L-[3H] glutamate to multiple sites on rat hippocampal synaptic membranes. Quisqualate (5 .mu. M) or DL-2-amino-4-phosphonobutyrate (2-APB) (300 .mu. M) was used to discriminate 2 classes of binding sites. Saturation isotherms and displacement surves constructed under different ionic conditions suggested that the effects of Cl- and Ca2= could best be explained by postulating the existence of 3 major binding site populations in this preparation rather than 2. The binding of L-glutamate to Glu A sites exhibits an absolute dependence on Cl- and Ca2+ markedly increases the maximum density of these sites. Glu A sites bind quisqualate and 2-APB with relatively high affinity. Cl- (47 m M) more than doubles the maximum density of Glu B sites, but Ca2+ appears to have no effect. Glu B sites can be discriminated from the other classes by their relatively low affinity for quisqualate and 2-APB. The Glu B population is probably heterogeneous. The novel Glu C population can be virtually selectively labeled by exposing 2-APB-sensitive binding sites to radioligand in Tris-HOAc buffer with Ca2+. Binding of L-[3H] glutamate to these sites is enhanced by both Cl- and Ca2+, but requires neither ion. Ca2+ appears to increase both affinity of Glu C sites for L-glutamate and their maximum binding sites density. In the presence of Ca2+ and Cl-, Glu C sites bind the radioligand with micromolar affinity (Kd .simeq. 2 .mu. M) and high capacity (Bmax .simeq. 160 pmol/mg protein). The structural specificity of these binding sites closely resembles that of Glu A sites, but these sites bind excitants with about an order of magnitude lower affinity. The Glu C binding sites exhibit characteristics compatible with a synaptic receptor function in the hippocampal formation.