Comparison of cysteine string protein (Csp) and mutant alpha-SNAP overexpression reveals a role for Csp in late steps of membrane fusion in dense-core granule exocytosis in adrenal chromaffin cells

Assembly of the SNARE complex and its disassembly caused by the action of s oluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) an d NSF is crucial for the maintenance of vesicular traffic, including fusion of regulated exocytotic vesicles. Various other proteins may also have imp ortant roles in the processes leading to membrane fusion via interaction wi th the SNARE proteins, including the secretory vesicle cysteine string prot ein (Csp). Here we have examined the effect of overexpression of a dominant negative alpha-SNAP mutant or Csp on exocytosis of dense-core granules in single chromaffin cells monitored using amperometry to detect released cate cholamine. Exocytosis of trans-Golgi network (TGN)-derived dense-core granu les was substantially inhibited by expression of alpha-SNAP(L294A). The amp litude and characteristics of the individual release events were unaffected by expression of alpha-SNAP(L294A), consistent with an essential role for alpha-SNAP in early steps of priming but not in the fusion process. In cont rast, Csp overexpression, which also inhibited the extent of exocytosis, al so modified the kinetics of the individual release events seen as an increa se in the rise time and a broadening of the residual amperometric spikes in Csp-transfected cells. These results suggest that unlike alpha-SNAP, Csp p lays a key role in the protein interactions close to the fusion process or fusion pore opening during Ca2+-regulated exocytosis.