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  • Title: Differentiation of the Ca2+-stimulated binding from the Cl- -dependent binding of [3H]glutamate in synaptic membranes from rat brain.
    Author: Ogita K, Yoneda Y.
    Journal: Neurosci Res; 1986 Dec; 4(2):129-42. PubMed ID: 2880324.
    Abstract:
    The effect of Ca2+ as well as Cl- ions on [3H]glutamate (Glu) binding was re-examined using rat brain synaptic membranes frozen at -80 degrees C in 0.32 M sucrose. The inclusion of 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate disclosed the Cl- -dependent binding or Ca2+-stimulated binding even at 2 min after the initiation of incubation at 30 degrees C and each binding reached a plateau within 30 min. In contrast, the binding reached its maximal value within 10 min followed by a progressive decline up to 60 min in the presence of 100 mM sodium acetate. Scatchard analysis revealed that Cl- as well as Cl-/Ca2+ ions invariably caused a significant increment of the number of binding sites without altering their affinity, whereas Na+ ions induced a prominent increment of the density of binding sites with a concomitant lowering of their affinity. DL-2-Amino-4-phosphonobutyric acid selectively abolished the Cl- -dependent and Ca2+-stimulated bindings without significantly affecting the basal or Na+-dependent binding. Quisqualic acid induced a profound inhibition of both Cl- -dependent and Ca2+-stimulated bindings, to a significantly greater extent than that of the basal and Na+-dependent bindings. D-Aspartic acid exhibited a potent inhibition of the Na+-dependent binding with a significantly less potent displacement of the basal, Cl- -dependent and Ca2+-stimulated bindings. An inhibitor of anion transport, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), not only eliminated the Cl- -dependent binding, but also completely abolished the Ca2+-stimulated binding. Scatchard analysis revealed that DIDS (0.1 mM) prevented the Cl- - and Cl-/Ca2+-induced increment of the density of binding sites with no significant change of their affinity. Pretreatment of the membranes with hydrophilic SH-reactive agents such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) invariably resulted in a more sensitive inhibition of the Ca2+-stimulated binding than that of the Cl- -dependent binding, while hydrophobic reagent p-chloromercuribenzoic acid produced a similarly potent elimination of the Cl- -dependent and Ca2+-stimulated bindings. Calcium-stimulated binding was also found to be sensitively diminished by dithiothreitol and dithioerythritol as compared with the Cl- -dependent binding. In vitro addition of L-ascorbic acid (10(-6)-10(-3) M) attenuated the Ca2+-stimulated binding to a significantly greater extent than the inhibition of the Cl- -dependent binding.(ABSTRACT TRUNCATED AT 400 WORDS)
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