Binding sites for L-[3H]glutamate on hippocampal synaptic membranes: three populations differentially affected by chloride and calcium ions.
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 microM) or DL-2-amino-4-phosphonobutyrate (2-APB) (300 microM) was used to discriminate two previously identified classes of binding sites. Saturation isotherms and displacement curves constructed under different ionic conditions suggested that the effects of Cl- and Ca2+ could best be explained by postulating the existence of three major binding site populations in this preparation rather than two. 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 mM) 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. There is reason to think that the Glu B population is 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 the affinity of Glu C sites for L-glutamate and their maximum binding site density. In the presence of Ca2+ and Cl-, Glu C sites bind the radioligand with micromolar affinity (KD approximately 2 microM) and high capacity (Bmax approximately 160 pmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)
Nadler
J V
JV
Wang
A
A
Werling
L L
LL
D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002118:Calcium / Q000494:pharmacology*; D002712:Chlorides / Q000494:pharmacology*; D005260:Female; D006624:Hippocampus / Q000032:analysis*; D066298:In Vitro Techniques; D051381:Rats; D011919:Rats, Inbred Strains; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000032:analysis*; D014316:Tritium
D002021:Buffers; D002712:Chlorides; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D014316:Tritium; D002118:Calcium