192 nature neuroscience • volume 1 no 3 • july 1998 articles Functional studies on secretory cells have supplied ample evi- dence that synaptic and other secretory vesicles can exist in distinct functional states 1–4 . Typically, only a fraction of all vesicles of a secretory cell can be released in a certain time window, independent of the strength of a given stimulus. These vesicles in a specific release-ready state are termed a ‘pool’. The question arises whether pools defined in this or other ways can be associated with well defined macromolecu- lar complexes of synaptic proteins, which have recently been characterized biochemically 5–7 . Some of the proteins of these complexes, such as SNAP-25, synaptobrevin and syntaxin, are the targets of very specific proteolytic neurotoxins, which cleave the proteins at unique sites 8–10 . These proteins are high- ly susceptible to toxin attack in monomeric form, whereas they are protected to various degrees in macromolecular complex- es 11 . This property and differences between the toxins’ actions may allow us to link certain functional pools to some of the biochemically defined complexes by their toxin susceptibili- ty. Indeed, different kinetic components are impaired differ- entially by toxins 12,13 . Also, botulinum neurotoxin type A may be special among the clostridial neurotoxins, as its action in some preparations can be overcome by strong stimuli 14,15 . For a precise dissociation of functional steps and their cor- relation with molecular data, it would be desirable to study exocytosis at fast resolution, as the final steps in the neurose- cretory pathway are known to be fast, and most likely neuro- toxins act at later steps. We therefore studied catecholamine secretion from bovine chromaffin cells using capacitance mea- surement as a fast assay of exocytosis combined with flash photolysis of caged calcium as a fast and strong stimulus. We characterized two kinetic components that were differentially affected by clostridial neurotoxins. We compared the capaci- tance signal with simultaneously measured catecholamine release, and thereby identified a third very prominent kinet- ic component, which apparently was not related to cate- cholamine release, and which was not affected by toxins. Together, these data suggest possible relationships between late steps in the release process and their biochemically defined molecular counterparts. Results MULTIPLE COMPONENTS OF EXOCYTOSIS IN CHROMAFFIN CELLS To study the kinetic components of secretion, we employed fast-resolution capacitance measurements to estimate the secre- tory response after spatially homogeneous elevation of inter- nal calcium concentration ([Ca 2+ ] i ) by photorelease of caged calcium, nitrophenyl-EGTA. Membrane capacitance (Cm) is proportional to the surface area of the cell, and it increases when secretory vesicles fuse with the plasma membrane 16,17 . In most of our measurements, we used the following protocol ( Fig. 1a). After ten minutes of whole-cell dialysis with either toxin-free or toxin-containing pipette solutions, we first gave low-intensity flashes (usually two) to generate small [Ca 2+ ] i jumps to about 20 μM. This readily elicited secretory respons- es ( Fig. 1b). When there was a clear indication that secretion was depressed, we then gave strong flashes to elevate [Ca 2+ ] i to higher values (over 100 μM). Intervals between the flashes were 120 seconds. In response to the first flash, there was always a robust Cm increase with two clearly distinct phases, which we call the exocytic burst 18 and the slow component. Our Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity Tao Xu 1 , Thomas Binz 2 , Heiner Niemann 2 and Erwin Neher 1 1 Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany 2 Department of Biochemistry, Medizinische Hochschule, D-30625, Hannover, Germany Correspondence should be addressed to T.X. (txu@gwdg.de) The secretion of synaptic and other vesicles is a complex process involving multiple steps. M any mol- ecular 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 exo- cytosis, 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. © 1998 Nature America Inc. • http://neurosci.nature.com © 1998 Nature America Inc. • http://neurosci.nature.com