Characterization of Two [³H]Glutamate Binding Sites in Rat Hippocampal Membranes

Abstract

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5-5.0 mM), whereas higher concentrations resulted in a dose-dependent stimulation of binding (in the range 10-150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable, exhibiting dissociation constants at 30 degrees C of 750 nM and 2.4 microM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+-independent and Na+-dependent binding and suggested that the former sites were related to post-synaptic glutamate receptors, whereas the latter were related to high-affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+-dependent binding site, which paralleled that of the high-affinity glutamate uptake; the Na+-independent binding exhibited less regional variation.

Classical conditioning of the rabbit eyelid response increases glutamate receptor binding in hippocampal synaptic membranes.

“…Rabbit hippocampal synaptic membranes were found to possess a high-affinity, Na'-independent, [3H]glutamate-binding site with kinetic, ionic, and pharmacological properties similar to those sites previously characterized in rat hippocampus (11,16 (17) and under some conditions in rat hippocampus (18), and these sites have also been related to a physiological postsynaptic receptor.…”

The blocking action of baclofen on excitatory transmission in the rat hippocampal slice

“…Hippocampal membranes were prepared and immediately assayed for r3H]glutamate binding in the presence or absence of calcium as previously described (Baudry and Lynch, 1981a). High affinity [3H] glutamate uptake was measured on crude mitochondrial fractions according to the procedure of Sandoval et al (1978) as described (Baudry and Lynch, 1981a).…”

Effects of the paratemnus elongatus pseudoscorpion venom in the uptake and binding of the L‐glutamate and GABA from rat cerebral cortex

“…We used the method of Baudry and Lynch [35], Zukin et al [36], and modified by Carolino et al [37]. The binding assays were initiated with the addition of the following into Eppendorf tubes: 50 nM 3 H-Lglu (NEN, 21.6 Ci/mmol), or 3 nM 3 H-GABA in an ice bath (NEN, 65 Ci/mmol), and Paratemnus crude venom (2.0-0.028 U, for both L-glu and GABA).…”