Characterization of Na +-dependent binding sites of [ 3H]glutamate in synaptic membranes from rat brain

Abstract

Some biochemical characteristics of Na +-dependent binding of [ 3H] l-glutamic acid (Glu) were studied using crude synaptic membrane preparations from the rat brain as compared with Na +-independent binding. In vitro addition of sodium chloride (1–100 mM) exhibited a significant enhancement of [ 3H]Glu binding to synaptic membranes in a concentration-dependent manner independent of the incubation temperature employed (2 or 30 °C). In contrast, sodium acetate elicited a concentration-dependent augmentation of the binding at 2 °C to a significantly greater extent than that found at 30 °C. It was found that the binding found in the presence of 100 mM sodium acetate reached its maximal value within 10 min of incubation followed by a rapid decline up to 60 min at 30 °C, while gradually increasing up to 60 min at 2 °C. The Na +-independent basal binding was significantly activated by the alteration of incubation temperature from 2 to 30 °C and reached equilibrium within 10 min of incubation at both incubation temperatures. The Na +-dependent binding was more promptly attenuated by the addition of excess of non-radiactive Glu (1 mM) at 30 °C than that at 2 °C, whereas the Na +-independent binding was greatly suppressed by the addition at 2 °C in comparison with that at 30 °C. Quisqualic acid induced a considerably less-potent inhibition of the Na +-dependent binding than that of the Na +-independent binding. Neither N-methyl- l-aspartic acid nor kainic acid had such a significant effect on each binding. d-Aspartic acid exerted a significant elimination of the Na +-dependent binding in a concentration-dependent manner without significantly affecting the Na +-independent binding. In vitro addition of sodium acetate (1–100 mM) exhibited a concentration-dependent suppression of [ 3H]Glu binding in the presence of d-aspartate. Pretreatment of synaptic membranes with various sulphhydryl reagents such asp-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM) and5, 5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) invariably resulted in a significant deterioration of the Na +-independent binding in a concentration-dependent manner, whereas the Na +-dependent binding was significantly abolished exclusively by 1 mM PCMB. These results suggest that the Na +-dependent binding sites may be distinctly different from the Na +-independent binding sites in nature and the former sites may be closely related to the Na +-dependent sites of Glu uptake with a high affinity for this putative neurotransmitter.