L‐[³H]Glutamate Binding to Hippocampal Synaptic Membranes: Two Binding Sites Discriminated by Their Differing Affinities for Quisqualate

Abstract

The excitatory glutamate analogs quisqualate and ibotenate were employed to distinguish multiple binding sites for L-[3H]glutamate on freshly prepared hippocampal synaptic membranes. The fraction of bound radioligand that was displaceable by 5 microM quisqualate was termed GLU A binding. That which persisted in the presence of 5 microM quisqualate, but was displaceable by 100 microM ibotenate, was termed GLU B binding. GLU A binding equilibrated within 5 min and remained unchanged for up to 80 min. GLU B binding appeared to equilibrate at least as rapidly, but incubation with ligand unmasked latent binding sites. Saturation binding curves were best fitted by single exponentials, which yielded KD values of about 200 nM (GLU A) and 1 microM (GLU B). On the average, GLU B binding sites were about twice as abundant in these membranes as were GLU A sites. Rapid freezing of the membranes, followed by storage at -26 degrees C and rapid thawing markedly diminished GLU A binding, but nearly tripled GLU B binding. Both site bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with about 10 times the affinity of L-aspartate and discriminated poorly between L- and D-aspartate. In contrast, the GLU B site bound L-aspartate with an affinity similar to that for L-glutamate, and with an order-of-magnitude greater affinity than D-aspartate. The structural specificities of the GLU A and GLU B binding sites suggest that these sites may correspond to receptors on hippocampal pyramidal cell dendrites that are activated by iontophoretically applied L-glutamate.

Anion transport blockers inhibit dl‐2‐amino‐4‐phosphonobutyrate responses induced by quisqualate in the rat cerebral cortex

“…The role these transport/L-glutamate/AP4 sites play in the induction of AP4 responses remains unresolved, primarily owing to the problems of poor reversibility for DIDS, and the antagonism of non-NMDA receptors by SITS. However, the proposed sequestration of Quis via these sites (Zaczek et al, 1987b) is supported in the present study by the fact that responses to Quis rather than AMPA are relatively selectively enhanced by anion transport blockers, consistent with the >60 fold greater potency of Quis over AMPA to displace binding (Butcher et al, 1983;Werling et al, 1983). This is circumstantial evidence indicating that the exchange hypothesis is possible, but there is no direct evidence that Quis has indeed been sequestrated by the cortical tissue, although this has been demonstrated in the hippocampus (Harrison & Kilpatrick, 1991).…”

“…The flow rate was approximately 2 ml min-' and the temperature maintained at 21-23°C. All (Simon et al, 1976;Butcher et al, 1983;Werling et al, 1983;Kessler et al, 1987;Murphy et al, 1987;Pullan et al, 1987;Olverman et al, 1988). L-a-Aminoadipate (AA), L-cysteine (Cys) and L-cystathionine (CTN), fulfilled this criterion, although the first two are weakly active at NMDA receptor binding sites.…”

“…This could explain the relatively small responses obtained for L-o-AA in Figure 3, as the L-x-AA applied before the Quis could be blocking the induction process. In addition, since the same concentration of L-x-AA and DL-AP4 were used in these experiments, and these compounds are almost equipotent as displacers of Cl--dependent L-glutamate/AP4 binding sites (Butcher et al, 1983;Werling et al, 1983), the data would suggest that it is unlikely that these sites are involved in the induction of responses. However, without either comparing the DL-AP4 concentration-effect curve for control induction with those for induction in the presence of DL-AP4 or L-a-AA, or using different concentrations of DL-AP4 and L-o-AA during the induction process, it is impossible to measure the difference in potency with respect to the inhibition of induction.…”

“…However, since the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, but not NMDA antagonists, blocks the L-AP4 response in the cortex (Sheardown, 1988;Lodge & Zeman, 1989), the production of these responses would appear to be dependent on the activation of non-NMDA rather than NMDA receptors. and DL-[3H]-AP4 binding have very similar pharmacological profiles, indicating that they probably represent the same site (Werling et al, 1983). These binding correlates have been suggested to represent Cl--dependent L-glutamate sequestration (Pin et al, 1984), mediated via an anion transport mechanism sensitive to 4,4'-diisothiocyanatostilbene-2-2'-disulphonic acid (DIDS) (Recasens et al, 1987;Zaczek et al, 1987a).…”

l‐Glutamate diethyl ester and deaminated analogues as excitatory amino acid antagonists in rat cerebral cortex

“…Since responses to AMPA are unaffected by GDEE HCl, the potentiation of responses to Quis is also not due to a direct action on the AMPA/Quis receptor. is some evidence that GDEE displaces one of the components of Cl--dependent [3H]-L-glutamate binding (Werling et al, 1983), which probably represents Cl--dependent L-glutamate uptake, in view of its comparable pharmacological profile to [3H]-AP4 binding (Butcher et al, 1983) and its sensitivity to freeze-thawing. Since Quis is much more potent than AMPA in displacing this binding and Quis is a known substrate of Cl--dependent uptake (Zaczek et al, 1987), a block of this uptake would explain these results.…”