scholarly journals Distinct effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis.

1995 ◽  
Vol 130 (5) ◽  
pp. 1063-1070 ◽  
Author(s):  
L H Chamberlain ◽  
D Roth ◽  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Membrane Fusion ◽  
Chromaffin Cells ◽  
Time Course ◽  
Mechanisms Of Action ◽  
Catecholamine Secretion ◽  
Adrenal Chromaffin Cells ◽  
Regulated Exocytosis ◽  
Distinct Stage ◽  
Adrenal Chromaffin

We have used stage-specific assays for MgATP-dependent priming and for Ca(2+)-activated triggering in the absence of free MgATP to examine the effects of alpha-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca(2+)-dependent increase in catecholamine secretion. Both alpha-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of alpha-SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca(2+)-dependency, time course and extent of stimulation and their effects were additive. alpha-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that alpha-SNAP acts in an early MgATP-requiring stage and not in the late Ca(2+)-triggered steps immediately prior to membrane fusion as previously suggested.

Control of membrane fusion dynamics during regulated exocytosis

2001 ◽  
Vol 29 (4) ◽  
pp. 467-472 ◽  
Author(s):  
R. D. Burgoyne ◽  
R. J. Fisher ◽  
M. E. Graham ◽  
L. P. Haynes ◽  
A. Morgan
Keyword(s):  
Membrane Fusion ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
General Strategy ◽  
Adrenal Chromaffin Cells ◽  
Regulated Exocytosis ◽  
Cysteine String Protein ◽  
Adrenal Chromaffin ◽  
Pore Dynamics

The study of regulated exocytosis uniquely allows the direct measurement of intracellular membrane fusion events in real time. We have exploited this to examine factors that regulate not only the extent but also the dynamics of single fusion/release events. The general strategy used has been to assess exocytosis in transiently transfected PC12 or adrenal chromaffin cells. We aimed to design mutant constructs based on in vitro biochemistry, in some cases informed by knowledge of protein structure. Using this approach we have demonstrated an inhibitory role for the putative Rab3 effector Noc2 that requires interaction with Rab3. Using carbon-fibre amperometry on adrenal chromaffin cells, we have demonstrated regulation of the kinetics of single granule release events consistent with changes in fusion pore dynamics and switches between full fusion and ‘kiss-and-run’ fusion. These studies have demonstrated a late role for cysteine string protein in exocytosis. In addition, they have focused attention on a key role for Munc18 in the regulation of post-fusion events that affect fusion pore dynamics.

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