Methods for analysis of Ca(2+)/H(+) antiport activity in synaptic vesicles isolated from sheep brain cortex.

The involvement of Ca(2+)-storage organelles in the modulation of synaptic transmission is well-established [M.K. Bennett, Ca(2+) and the regulation of neurotransmitter secretion, Curr. Opin. Neurobiol. 7 (1997) 316-322 [1]; M.J. Berridge, Neuronal calcium signaling, Neuron 21 (1998) 13-26 [2]; Ph. Fossier, L. Tauc, G. Baux, Calcium transients and neurotransmitter release at an identified synapse, Trends Neurosci. 22 (1999) 161-166 [7] ]. Various Ca(2+) sequestering reservoirs (mitochondria, endoplasmic reticulum and synaptic vesicles) have been reported at the level of brain nerve terminals [P. Kostyuk, A. Verkhratsky, Calcium stores in neurons and glia, Neuroscience 63 (1994) 381-404 [18]; V. Mizuhira, H. Hasegawa, Microwave fixation and localization of calcium in synaptic terminals using X-ray microanalysis and electron energy loss spectroscopy imaging, Brain Res. Bull. 43 (1997) 53-58 [21]; A. Parducz, Y. Dunant, Transient increase of calcium in synaptic vesicles after stimulation, Neuroscience 52 (1993) 27-33 [23]; O.H. Petersen, Can Ca(2+) be released from secretory granules or synaptic vesicles?, Trends Neurosci. 19 (1996) 411-413 [24] ]. However, the knowledge of the specific contribution of each compartment for spatial and temporal control of the cytoplasmic Ca(2+) concentration requires detailed characterization of the Ca(2+) uptake and Ca(2+) release mechanisms by the distinct intracellular stores. In this work, we described rapid and simple experimental procedures for analysis of a Ca(2+)/H(+) antiport system that transport Ca(2+) into synaptic vesicles at expenses of the energy of a DeltapH generated either by activity of the proton pump or by a pH jumping of the vesicles passively loaded with protons. This secondary active Ca(2+) transport system requires high Ca(2+)100 microM) for activation, it is dependent on the chemical component (DeltapH) of the proton electrochemical gradient across the synaptic vesicle membrane and its selectivity is essentially determined by the size of the transported cation [P.P. Gonçalves, S.M. Meireles, C. Gravato, M.G. P. Vale, Ca(2+)-H(+)-Antiport activity in synaptic vesicles isolated from sheep brain cortex, Neurosci. Lett. 247 (1998) 87-90 [10]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Ionic selectivity of the Ca(2+)/H(+) antiport in synaptic vesicles of sheep brain cortex, Mol. Brain Res. 67 (1999) 283-291 [11]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Synaptic vesicle Ca(2+)/H(+) antiport: dependence on the proton electrochemical gradient, Mol. Brain Res. 71 (1999) 178-184 [12] ]. The protocols described here allow to ascertain the characteristics of the Ca(2+)/H(+) antiport in synaptic vesicles and, therefore, may be useful for clarification of the physiological role of synaptic vesicles in fast buffering of Ca(2+) at various sites of the neurotransmission machinery.