Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity (original) (raw)
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‘Full fusion’ is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells
Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2017
Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering (‘Kiss-and-Run’ events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane—a stage called ‘full fusion’. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of ‘full fusion’.
Docked Secretory Vesicles Undergo Ca2+-activated Exocytosis in a Cell-free System
Journal of Biological Chemistry, 1997
The Ca 2؉-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca 2؉-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca 2؉ (EC 50 ϳ 3 M) and exhibited a dependence upon MgATP and cytosol. Ca 2؉-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca 2؉-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca 2؉-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway.
Cytosolic calcium facilitates release of secretory products after exocytotic vesicle fusion
FEBS Letters, 1995
We monitored single vesicle exocytosis by simultaneous measurements of cell membrane capacitance as an indicator of fusion and amperometric detection of serotonin release. We show here that vesicle-plasma membrane fusion in rat mast cell granules is followed by a variable, exponentially distributed, delay before bulk release. This delay reflects the time required for the expansion of the exocytotic fusion pore, lasting, on average, 231 ms in resting cytosolic calcium, [Ca2+L (50 nM). In the presence of [Ca2+]i in the low micromollar range, the lag between fusion and release was reduced to 123 ms. The characteristics of the amperometric signals were unchanged by [Ca2+]i. These resuits show a novel site of regulation in the exocytotic process, the fusion pore, which may represent a different mechanism facilitating transmitter release.
Journal of Neurochemistry, 2003
The impact of syntaxin and SNAP-25 cleavage on [ 3 H]noradrenaline ([ 3 H]NA) and [ 3 H]dopamine ([ 3 H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca 2+ -dependent K + -induced [ 3 H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [ 3 H]NA and [ 3 H]DA were differentially affected: both toxins blocked the former, dependent on external Ca 2+ , but not the latter, internal Ca 2+ -dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [ 3 H]catecholamine release.
Toxicology, 2002
Inhibition of regulated exocytosis by botulinum toxins type A and B was studied in chromaffin cells. Both virtually abolished catecholamine release triggered from intact cells by depolarising stimuli, whereas the blockade by type A, but not B, was only partial after cell permeabilisation and direct stimulation of exocytosis by Ca 2' . Botulinum toxin A did not alter the [Ca 2' ]-dependency of exocytosis in permeabilised cells but, rather, proportionally reduced the amount of release at each concentration tested. Likewise, this toxin decreased the extents of Ca 2' -induced structural changes in SNAP-25, synaptobrevin and syntaxin (known collectively as SNAREs), whilst leaving their [Ca 2' ]-sensitivity unaltered. Thus, botulinum toxin A does not reduce the Ca 2' -sensitivity of the exocytosis sensor, but hinders transmission of the signal to the SNAREs which mediate fusion. #
European Journal of Biochemistry, 1996
Botulinum neurotoxin (BoNT) types A and B are ZnZ '-requiring endoproteases which potently block neurotransmitter release by cleavage of a 25-kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, respectively. Synaptobrevin is important for the exocytosis of catecholamines from dense-core granules and evidence is presented here for the involvement of SNAP-25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by BaZ+, by elevated K+ concentrations or by nicotine was prevented by each toxin. A very good correlation was observed between the extents of SNAP-25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT/A or BoNT/B, respectively, which indicates the importance of SNAP-25 and synaptobrevin in regulated exocytosis. Despite truncation of almost the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20 pM CaZ+ (and to a lesser extent by 1 mM BaZ+) following permeabilisation. Addition of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg . ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+-stimulated or Ba*+-stirnulated BoNT/A-resistant secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of the ability of BoNTE to abolish secretion from permeabilised cells and to delete 26 amino acids from the C-terminus of SNAP-25, it can be deduced that cleavage of only nine residues by BoNT/A does not prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP-25 with other proteins involved in regulated exocytosis.
FEBS Letters, 1995
SNAP-25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicleplasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP-25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti-SNAP-25 monoclonal antibody to show that SNAP-25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells.
Brain Research Reviews, 2000
Neuroendocrine cells display a similar calcium dependence of release as synapses but a strongly different organization of channels and vesicles. Biophysical and biochemical properties of large dense core vesicle release in neuroendocrine cells suggest that vesicles and channels are dissociated by a distance of 100-300 nm. This distinctive organization relates to the sensitivity of the release process to mobile calcium buffers, the resulting relationship between calcium influx and release and the modulatory mechanisms regulating the efficiency of excitation-release coupling. At distances of 100-300 nm, calcium buffers determine the calcium concentration close to the vesicle. Notably, the concentration and diffusion rate of mobile buffers affect the efficacy of release, but local saturation of buffers, possibly enhanced by diffusion barriers, may limit their effects. Buffer conditions may result in a linear relationship between calcium influx and exocytosis, in spite of the third or fourth power relation between intracellular calcium concentration and release. Modulation of excitation-secretion coupling not only concerns the calcium channels, but also the secretory process. Transmitter regulation mediated by cAMP and PKA, as well as use-dependent regulation involving calcium, primarily stimulates filling of the releasable pool. In addition, direct effects of cAMP on the probability of release have been reported. One mechanism to achieve increased release probability is to decrease the distance between channels and vesicles. GTP may stimulate release independently from calcium. Thus, while in most cases primary inputs triggering these pathways await identification, it is evident that large dense core vesicle release is a highly controlled and flexible process.
The Journal of biological chemistry, 1997
Excitation-secretion uncoupling peptides (ESUPs) are inhibitors of Ca2+-dependent exocytosis in neural and endocrine cells. Their mechanism of action, however, remains elusive. We report that ESUP-A, a 20-mer peptide patterned after the C terminus of SNAP-25 (synaptosomal associated protein of 25 kDa) and containing the cleavage sequence for botulinum neurotoxin A (BoNT A), abrogates the slow, ATP-dependent component of the exocytotic pathway, without affecting the fast, ATP-independent, Ca2+-mediated fusion event. Ultrastructural analysis indicates that ESUP-A induces a drastic accumulation of dense-core vesicles near the plasma membrane, mimicking the effect of BoNT A. Together, these findings argue in favor of the notion that ESUP-A inhibits ATP-primed exocytosis by blocking vesicle docking. Identification of blocking peptides which mimic sequences that bind to complementary partner domains on interacting proteins of the exocytotic machinery provides new pharmacological tools to ...
Biochemistry, 1997
Types A and E botulinum neurotoxin (BoNT) are Zn 2+ -requiring endoproteases which cleave nine and twenty-six residues, respectively, from the C-terminus of synaptosomal-associated protein of M r ) 25 kDa (SNAP-25). Involvement of SNAP-25 in the exocytosis of large dense-core vesicles in bovine adrenochromaffin cells was examined by measuring cleavage of SNAP-25 in relation to the levels of Ca 2+ -evoked catecholamine release from cells exposed to BoNT/A or /E, either before or after permeabilization. The dose-dependency of inhibition of exocytosis correlated closely with the extents of SNAP-25 cleavage in cells permeabilized and then treated with BoNT/E. In intact cells exposed to 66 nM BoNT/A, virtually all of the SNAP-25 was truncated, accompanied by a near-complete inhibition of exocytosis; however, after their permeabilization a significant level of secretion was recorded upon Ca 2+stimulation. Importantly, this BoNT/A-resistant release from the permeabilized cells was dramatically lowered by subsequently adding BoNT/E, which further truncated the SNAP-25 fragment (lacking the C-terminal nine residues) that had been produced earlier by BoNT/A. Moreover, anti-SNAP-25 IgG decreased the BoNT/A-insensitive exocytosis. When permeabilized cells were exposed to either neurotoxin, both blocked MgATP-dependent secretion but only BoNT/E attenuated the energy-independent phase. These distinct inhibitory effects of the two neurotoxins demonstrate that residues 197-205 at the C-terminus of SNAP-25 are absolutely essential for exocytosis from intact cells whereas even after their removal a significant proportion of the exocytotic response can be elicited from permeabilized cells, but this is reliant on amino acids 180-196. Moreover, the latter but not residues 197-205 are implicated in a late, MgATP-independent step of exocytosis, which is blocked by BoNT/E but nonsusceptible to BoNT/A.