Biochemical and functional studies of cortical vesicle fusion: The SNARE complex and Ca2+ sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exoc ytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV-PM fusion). He re we show that Sr2+ and Ba2+ also trigger CV-CV fusion, and agents affecti ng different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggere d 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 exo cytosis. Both < n >(Max) and the Ca2+ sensitivity of fusion complex activat ion were comparable to that determined for CV-PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membra ne fusion (CV-CV and CV-PM) and the SNARE complex, Fusion and complex disru ption have different sensitivities to Ca2+ Sr2+, and Ba2+, the complex rema ins Ca2+- sensitive on fusion-incompetent CV, and disruption does not corre late with the quantified activation of fusion complexes. Under conditions w hich disrupt the SNARE complex, CV on the PM remain docked and fusion compe tent, and isolated CV still dock and fuse, but with a markedly reduced Ca2 sensitivity. Thus, in this system, neither the formation, presence, nor di sruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the unive rsal minimal fusion machine for intracellular fusion. We suggest that: this complex modulates the Ca2+ sensitivity of fusion.