Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes

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Abstract
Synaptosomal membranes accumulate 3–6 times more Ca2+ in the presence of ATP (50–1000 μM) than basal Ca2+ accumulation (-ATP). The location of this Ca2+ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca2+ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 μg/ml), sodium azide (100 μM) and dinitrophenol (100 μM) differentiate mitochondrial from nonmitochondrial Ca2+ accumulation under conditions that are [Ca2+]- and ATP-dependent. In the presence of low concentrations of ATP (free 2+ (2.5 or 6.8 μM), Ca2+ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 μM), the Ca2+ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca free 2+ =2.5 μM. When Ca free 2+ is increased to 6.8 μM, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca2+ accumulating mechanisms in synaptosomal membranes depend on both [Ca2+] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca2+ uptake may be a viable mechanism for the regulation of synaptosomal Ca2+ levels.