Irreversibility and time course of calcium stimulated [3H]glutamate binding to rat hippocampal membranes

Previous studies have indicated that calcium ions induce an irreversible increase of [3H]glutamate binding to rat hippocampal membranes. The present study was intended to provide an estimate of the time-course for this effect. The calcium chelator EGTA and the proteinase inhibitor leupeptin were introduced at various times during the incubation of hippocampal membranes with calcium and the binding of [3H]glutamate subsequently assayed. Both compounds totally blocked the calcium-induced increase in binding when added at the beginning of the incubation period but produced progressively less inhibition when added after longer delays. This method indicates that calcium produces its maximal effect between 5 and 10 min of incubation, a time-course compatible with the hypothesis that calcium stimulates a membrane-bound proteinase which results in the unmasking of [3H]glutamate binding sites.