Fusion of isolated synaptic vesicles as a model of the terminal stage of regulated exocytosis

Abstract

In the study of membrane fusion, which is the terminal stage of exocytosis, we used a simplified model consisting of homotypic membranes of isolated synaptic vesicles (SV) obtained from the synaptosomal fraction of rat brain tissue. It was shown that fusion of SV develops in the presence of cytoplasmic proteins and 10−7 to 10−5 M Ca2+ ions. This conclusion was made based on changes in the intensity of fluorescence of a probe, R18. Calcium ions were found to be the most effective activators of the membrane fusion when the effects of bivalent cations, Ca2+, Sr2+, and Ba2+, were compared. ATP induced membrane fusion both in the presence and in the absence of Ca2+, and the effects of ATP and Ca2+ were additive. These findings allow us to believe that there are factors in the system containing SV and soluble proteins of synaptosomes, which initiate fusion of the membranes under the influence of not only Ca2+ but also ATP. The intensity of Ca2+-dependent fusion of SV dropped after trypsin treatment, i.e., proteolysis resulted in modulation of the sensitivity of vesicular proteins and/or a change in their capability of evoking membrane fusion. Monoclonal antibodies against synaptotagmin and synaptobrevin inhibited fusion of SV, but only partly. Our results support the concept that Ca2+-regulated membrane fusion is possible without the involvement of the entire SNARE complex.