Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca(2+) Sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca(2+) activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr(2+) and Ba(2+) also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca(2+), Sr(2+), and Ba(2+)-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>(Max), was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>(Max) and the Ca(2+) sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca(2+)-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca(2+), Sr(2+), and Ba(2+), the complex remains Ca(2+)- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca(2+) sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca(2+)-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca(2+) sensitivity of fusion.