Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity

Abstract

The secretion of synaptic and other vesicles is a complex process involving multiple steps. Many molecular components of the secretory apparatus have been identified, but how they relate to the different stages of vesicle release is not clear. We examined this issue in adrenal chromaffin cells, where capacitance measurements and amperometry allow us to measure vesicle fusion and hormone release simultaneously. Using flash photolysis of caged intracellular calcium to induce exocytosis, we observed three distinct kinetic components to vesicle fusion, of which only two are related to catecholamine release. Intracellular dialysis with botulinum neurotoxin E, D or C1 or tetanus-toxin light chains abolishes the catecholamine-related components, but leaves the third component untouched. Botulinum neurotoxin A, which removes nine amino acids from the carboxy(C)-terminal end of SNAP-25, does not eliminate catecholamine release completely, but slows down both catecholamine-related components. Thus we assign a dual role to SNAP-25 and suggest that its nine C-terminal amino acids are directly involved in coupling the calcium sensor to the final step in exocytosis.

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Figure 1: Multiple secretory components in response to different [Ca2+]i levels.
Figure 2: Kinetic analysis of multiple Cm components.
Figure 3: ATP dependence of secretion.
Figure 4: The effect of BoNT/E and BoNT/A on secretion.
Figure 5: The effect of BoNT/C1, BoNT/D and TeNT on secretion.
Figure 6: Secretion at high [Ca2+]i is sensitive to ATP but not to clostridial neurotoxins.
Figure 7: Hypothetical model for the kinetic steps leading to exocytosis.

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Acknowledgements

We would like to thank Dr. Ellis-Davies for samples of NP-EGTA, Drs. Corey Smith, Reinhard Jahn and Tobias Moser for feedback on the manuscript, and Frauke Friedlein and Michael Pilot for cell preparation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Nr. CHV-113/65/0) and from the European Community (Nr. CHRX-CT940500 ) to E.N. T.B. and H.N. were supported by the Fonds der chemischen Industrie and by the Deutsche Forschungsgemeinschaft (Nr. IIB2-Bi660/1-1).

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Correspondence to Tao Xu.

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Xu, T., Binz, T., Niemann, H. et al. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci 1, 192–200 (1998). https://doi.org/10.1038/642

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