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The Journal of Neuroscience, February 15, 2000, 20(4):1281-1289
Comparison of Cysteine String Protein (Csp) and Mutant
-SNAP Overexpression Reveals a Role for Csp in Late Steps of
Membrane Fusion in Dense-Core Granule Exocytosis in Adrenal
Chromaffin Cells
Margaret E.
Graham and
Robert D.
Burgoyne
The Physiological Laboratory, The University of Liverpool,
Liverpool L69 3BX, United Kingdom
Assembly of the SNARE complex and its disassembly caused by
the action of soluble N-ethylmaleimide-sensitive factor
(NSF) attachment protein (SNAP) and NSF is crucial for the
maintenance of vesicular traffic, including fusion of regulated
exocytotic vesicles. Various other proteins may also have important
roles in the processes leading to membrane fusion via interaction with the SNARE proteins, including the secretory vesicle cysteine string protein (Csp). Here we have examined the effect of overexpression of a
dominant negative -SNAP mutant or Csp on exocytosis of dense-core granules in single chromaffin cells monitored using amperometry to
detect released catecholamine. Exocytosis of trans-Golgi network (TGN)-derived dense-core granules was substantially inhibited by
expression of -SNAP(L294A). The amplitude and characteristics of the
individual release events were unaffected by expression of
-SNAP(L294A), consistent with an essential role for -SNAP in
early steps of priming but not in the fusion process. In contrast, Csp
overexpression, which also inhibited the extent of exocytosis, also
modified the kinetics of the individual release events seen as
an increase in the rise time and a broadening of the residual amperometric spikes in Csp-transfected cells. These results suggest that unlike -SNAP, Csp plays a key role in the protein interactions close to the fusion process or fusion pore opening during
Ca2+-regulated exocytosis.
Key words:
exocytosis; secretion; SNAP; NSF; Csp; amperometry
Copyright © 2000 Society for Neuroscience 0270-6474/00/2041281-09$05.00/0
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