Are Ca2+-dependent proteases really responsible for Cl(-)-dependent and Ca2+-stimulated binding of [3H]glutamate in rat brain?

The role of Ca2+ ions in [3H]glutamate binding was re-examined using synaptic membranous preparations obtained from the rat brain. In vitro addition (0.1-5 mM) of calcium chloride exhibited a profound enhancement of the binding in a temperature-dependent manner, whereas that of calcium acetate had no significant effect on the binding independently of the incubation temperature. Calcium acetate elicited a significantly additional stimulation of the Cl(-)-induced and temperature-dependent facilitation of the binding. The augmentation by these two ions was invariably eliminated by the addition of an antagonist for the anion channels including picrotoxinin as well as of inhibitors of anion transport such as ethacrynic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. L-Aspartate exerted a more potent inhibitory action on the Cl(-)-dependent binding and Cl(-)-dependent and Ca2+-stimulated binding, than D-aspartate. The latter two bindings were selectively abolished by an agonist (quisqualic acid) and an antagonist (DL-2-amino-4-phosphonobutyric acid) for central glutamate receptors, respectively. It was also found that pretreatment of the membranes with calcium acetate resulted in a complete abolishment of the Ca2+-stimulated binding with a concomitant stimulation of the Cl(-)-dependent binding, which invariably occurred independently of the preincubation temperature (2 or 30 degrees C). No significant alteration was detected in the basal binding following the latter pretreatment. None of various protease inhibitors such as leupeptin, antipain, chymostatin and pepstatin induced a significant alteration in the basal, Cl(-)-dependent, Ca2+-stimulated and Na+-dependent bindings of [3H]glutamate, respectively. These results suggest that Ca2+ ions may elicit their stimulatory action on the Cl(-)-dependent binding of [3H]glutamate even in the absence of Cl- ions added through the temperature-independent and apparently irreversible interaction with the anion transport carriers rather than the direct action on the binding sites of the ligand. The evidence presented here also suggests that the widely held view that Ca2+-dependent proteases are responsible for the exhibition of Cl(-)-dependent and Ca2+-stimulated binding of [3H]glutamate may need to be re-evaluated.