Fusion of neurotransmitter vesicles with target membrane is calcium-independent in a cell-free system

Abstract

In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2(+)-dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2(+)-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 30 degrees C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2(+)-dependent and -independent process: first, a Ca2(+)-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2(+)-independent processing to the terminal fusogen and subsequent Ca2(+)-independent fusion of the CGs with the PM.