(Received for publication, August 5, 1996, and in revised form, November 5, 1996)
From the Departamento de Neuroquímica, Instituto de Neurociencias and Facultad de Medicina, Universidad de Alicante, Apdo. 374, 03080 Alicante, Spain
From the Departamento de Histologia, Instituto de Neurociencias and Facultad de Medicina, Universidad de Alicante, Apdo. 374, 03080 Alicante, Spain
From the Department of Biology, University of California San Diego, La Jolla, California 92093-0366
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 dissect the molecular and mechanistic details of neurosecretion. Our findings may assist in developing ESUPs as substitute drugs to BoNTs for the treatment of spasmodic disorders.
Ca2+-dependent exocytosis is a highly regulated process in neural and endocrine cells. The molecular components and events involved in the exocytotic cascade are fast-emerging thanks to the convergence of genetic, biochemical, pharmacological, and biophysical approaches (1-7). The information accrued in recent years has led to the formulation of the SNAP1 receptors (SNARE) model to describe the final steps of the secretion cascade (1-9). The SNARE hypothesis distinguishes three distinct stages in the pathway, namely docking, priming, and fusion. Vesicle docking refers to the process by which cargo vesicles are targeted to the plasma membrane at the active zone, although they are not competent for Ca2+-triggered fusion (4). After docking, vesicles are activated by an ATP-dependent step known as vesicle priming. Primed vesicles are readily releasable in response to a transient Ca2+ elevation that triggers the fusion event.
Biochemically, docking is associated with the formation of a 7 S ternary complex involving the vesicle membrane protein synaptobrevin, also known as vesicle-associated membrane protein (VAMP), which is the vesicle SNARE (v-SNARE), and two plasma membrane proteins SNAP-25 and syntaxin, which comprise the target SNARE (t-SNARE) (2-6, 10-12). Vesicle priming is initiated by binding of the soluble proteins NSF (for N-ethylmaleimide-sensitive factor, an ATPase) and SNAPs (for soluble NSF attachment proteins, which are not related to SNAP-25) to the SNAP receptors (2-6). Specifically, VAMP binds to the SNAP-25-syntaxin heterodimer forming a ternary core complex that serves as a receptor for SNAPs and recruits NSF forming a 20 S complex (2-6, 9, 10, 13, 14). ATP hydrolysis by bound NSF energizes the secretory vesicles to the primed state, in which they are ready to fuse with the plasma membrane and release their content in response to the Ca2+ signal (1-7). The identification of an ATP-dependent, slow component in the final steps of the exocytotic cascade lends support to the SNARE hypothesis.
The discovery that Clostridial neurotoxins target-specific components of the v-SNARE and t-SNARE has contributed to our knowledge of the molecular entities comprising the exocytotic machinery, as well as the molecular events involved in Ca2+-mediated neuroexocytosis. Botulinum neurotoxins (BoNT) B, D, F, and G, and the structurally related tetanus toxin specifically cleave VAMP at different sites (15-17), whereas BoNT A and E cleave SNAP-25 at the C terminus (18, 19), and BoNT C cuts syntaxin and SNAP-25 (20, 21). Cleavage of any of these proteins prevents the formation of the core complex and abrogates Ca2+-triggered exocytosis (4).
Recently, synthetic peptides that mimic the amino acid sequence of segments from synaptotagmin (22, 23), SNAPs (24), synaptobrevin (25) and SNAP-25 (26, 27) were shown to be specific inhibitors of neurosecretion. These peptides, for which the term ESUP was coined to highlight their activity (26), are useful pharmacological tools to probe the functional role of distinct protein components in the secretory machinery, to dissect the contribution of specific domains in the protein-protein interactions that mediate the process and to identify steps in the exocytotic cascade. The use of this new set of reagents, however, remains limited because the mechanism underlying their inhibitory activity is unkonwn. Here, we characterize the molecular steps of the exocytotic process that are sensitive to the blocking activity of a 20-mer peptide that mimics the amino acid sequence of the C-terminal domain of SNAP-25 (SNAP-25-(187-206): SNKTRIDEANQRATKMLGSG; denoted as ESUP-A). We find that ESUP-A arrests Ca2+-dependent secretion from permeabilized chromaffin cells by inhibiting vesicle docking.
[3H]Noradrenaline was from Du Pont. t-butoxycarbonyl and Fmoc amino acids, with standard side chain protecting groups, were obtained from Applied Biosystems (Foster City, CA), NovaBio-chem (La Jolla, CA), or Peninsula Laboratories (Belmont, CA). Solvents, reagents, and resins for peptide synthesis were obtained from Applied Biosystems (Foster City, CA). All other reagents were of analytical grade from Sigma. BoNT A was a gift of B. R. DasGupta (University of Wisconsin, Madison, WI).
Peptide Synthesis and PurificationESUP-A (SNKTRIDEANQRATKMLGSG) and ESUP-ARDM (TDSSGREMIKANKQLANGTR) were synthesized by t-butoxycarbonyl or Fastmoc® Fmoc chemistries in an Applied Biosystems 431A automated solid-phase peptide synthesizer, and cleaved as described (26). Cleaved peptides were purified by reverse-phase HPLC with a VydaC C-18 semipreparative column. Samples of crude peptide (10-20 mg) were dissolved in 0.1% trifluoroacetic acid, applied to the column, and eluted with a linear gradient of 90% acetonitrile in 0.1% trifluoroacetic acid. Eluted peaks were monitored by absorbance measurements at 214 nm, pooled, and lyophilized. Peptide purity was assessed by reverse-phase HPLC in a VydaC C-18 analytical column.
Chromaffin Cell CulturesChromaffin cells were prepared from bovine adrenal glands by collagenase digestion and further separated from debris and erythrocytes by centrifugation on Percoll gradients as described (26, 28). Cells were maintained in monolayer cultures at a density of 625,000 cells/cm2 and were used between the 3rd and 6th day after plating. All the experiments were performed at 37 °C.
Determination of Catecholamine Release from Detergent-permeabilized Chromaffin CellsSecreted [3H]noradrenaline was determined in digitonin-permeabilized cells as described (26, 29). Briefly, cells were incubated with [3H]noradrenaline (1 µCi/ml) in Dulbecco's modified Eagle's medium supplemented with 0.56 mM ascorbic acid during 4 h. Thereafter, monolayers were washed four times with a Krebs/HEPES basal solution: 15 mM HEPES, pH 7.4, with 134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgCl2, 2.5 mM CaCl2, 0.56 mM ascorbic acid, and 11 mM glucose. Cell permeabilization was accomplished with 20 µM digitonin in 20 mM Pipes, pH 6.8 with 140 mM monosodium glutamate, 2 mM MgCl2, 2 mM Mg-ATP, and 5 mM EGTA. This incubation was carried out in the absence or presence of 100 µM ESUP-A, ESUP-ARDM, or 100 nM dithiothreitol (DTT)-reduced BoNT A as indicated. BoNT A was reduced with 10 mM DTT for 30 min at 37 °C. Following permeabilization, media were discarded, and cells were incubated for 10 additional min in digitonin-free medium in presence or absence of peptides. Basal secretion was measured in 5 mM EGTA, whereas stimulated secretion was measured in a medium containing 10 µM buffered Ca2+ solution. Media were collected, and released catecholamines as well as the total cell content were determined by liquid scintillation counting. Statistical significance was calculated using Student's t test with data from four or more independent experiments.
Electron Microscopy of Permeabilized Chromaffin CellsChromaffin cells (106 cells/well) were
permeabilized and peptide (100 µM) or DTT-reduced BoNT A
(100 nM) incubated as described above. Cells were collected
by low speed centrifugation (1,000 × g for 2 min) and
fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.05 M cacodylate buffer at pH 7.3 for 2 h. The pellets
were postfixed in 1.33% osmic acid during 1.5 h, in
s-collidine buffer for 2 h, dehydrated, and embedded in
Epon (30). Thin sections (~0.5 µm) were obtained using a Reichert
Ultracut-E ultramicrotome (Vienna, Austria). Electron microscopy was
performed using a transmission electron microscope (Zeiss EM-10,
Oberkochem, Germany).
Vesicle density distribution was estimated in digitized micrographs
using the NIH Image analysis program. For each chromaffin cell, the
distance between the dense core granules and the plasma membrane was
measured and binned using a bin width of 0.15 µm. To obtain a
distance distribution histogram (Fig. 5), the number of granules
occurring at given point was plotted as a function of the average
distance between the plasma membrane, defined at 0 µm, and the
nuclear membrane, at ~3 µm. Data are given as mean with number of
cells n 5 for each treatment; the number of vesicles (v) counted per cell was 240
v
290, and the
experimental error 10%
15%.
ESUP-A induces accumulation of secretory
vesicles in the active zone of chromaffin cells. The figure
depicts frequency distribution histograms of number of granules as a
function of distance to the plasma membrane for unstimulated cells
(left panels) and Ca2+-stimulated cells
(right panel). Vesicle distance to plasma membrane was
measured in digitized micrographs using the NIH Image program. Distance
was binned using a bin width of 0.15 µm within the boundaries of 0-3
µm, defined as the average distance between the plasma membrane (0 µm) and the nucleus (~3 µm). Distance distribution histograms
were obtained for unstimulated cells (Unstimulated) and
cells stimulated with 10 µM Ca2+
(Ca2+-stimulated). Four conditions
were analyzed: Control, untreated cells;
ESUP-ARDM, cells treated with 100 µM
ESUP-ARDM; ESUP-A, cells treated with 100 µM ESUP-A; and BoNT A, cells treated with 100 nM DTT-reduced BoNT A. Data are given as mean with number
of cells analyzed (n) 5. The number of vesicles measured per cell was 240
v
290. The
experimental error is 10%
15%. Solid lines
depict the best fit to a Lorentzian distribution with two components.
For control cells these components were characterized by the following
values: µ1 = 0.53 and µ2 = 2.3 µm, where µ is the mean distance to the plasma membrane. Different treatments
and Ca2+ stimulation did not affect the mean distance; the
modifications were in the relative frequency of the two vesicle
populations.
Ca2+-evoked exocytosis from permeabilized
chromaffin cells can be kinetically dissected in two distinct
components (31, 32) (Fig. 1): a fast component that may
be detected few seconds after Ca2+ application, lasts ~1
min, and releases ~40% of the total [3H]NE, followed
by a slow component that proceeds for ~15 min. This biphasic behavior
of catecholamine release has been associated with the presence of at
least two distinct pools of secretory granules at different stages of
the exocytotic cascade (3-6, 33). The fast component appears to
represent the release of neurotransmitter from a population of primed,
readily releasable vesicles, whereas the slow component corresponds to
a pool of docked vesicles that must undergo priming prior to fusion and other releasable pools of nondocked granules (3-6, 31, 33). We
investigated if ESUP-A targeted a specific step of the secretory pathway. As shown in Fig. 1, incubation of permeabilized chromaffin cells with 100 µM ESUP-A inhibited ~60% of
catecholamine release by primarily altering the slow phase of
secretion. These data suggest that the pool of primed vesicles is
insensitive to the action of ESUP-A.
To gain further insights on the specific secretory step inhibited by
ESUP-A, we attempted to dissociate the two distinct components of the
exocytotic process by performing a double Ca2+ pulse
secretion assay. The rationale considered that a short (2 min)
Ca2+ pulse would primarily deplete the pool of primed
vesicles, with minor effects on docked vesicles. A subsequent longer
(10 min) Ca2+ pulse would induce docking, priming, and
fusion of other vesicle pools. As illustrated in Fig. 2,
a short Ca2+ pulse triggered significant exocytosis of
[3H]NE from chromaffin cells that is highly insensitive
to 100 µM ESUP-A (~10% inhibition). In contrast,
ESUP-A blocked ~80% of the Ca2+-dependent
exocytosis elicited by the second, longer Ca2+ pulse. A
synthetic peptide with the same amino acid composition, yet random
sequence (ESUP-ARDM) blocked only ~10% of the
Ca2+-evoked neurosecretion, confirming that the ESUP-A
inhibitory activity is sequence-specific. Taken together, these data
suggest that ESUP-A inhibits Ca2+-evoked catecholamine
secretion by preventing vesicle docking or priming at the active zone,
yet do not argue in favor of one or the other.
ESUP-A Blocks the Energy-dependent Steps of Ca2+-mediated Exocytosis in Chromaffin Cells
Catecholamine release from chromaffin cells is an
energy-dependent process requiring ATP hydrolysis to induce
and stabilize the primed state of secretory vesicles before the
Ca2+-induced fusion event (1-7, 31, 32). Vesicle priming
is composed primarily of an ATP-dependent component
followed by a temperature-sensitive step that leads to the
Ca2+-induced exocytosis (32-34). As shown in Fig.
3, incubation of permeabilized chromaffin cells with 100 µM of ESUP-A attenuated ~60% of the
Ca2+-induced catecholamine secretion when 2 mM
ATP was present in the incubation and stimulation media (Fig. 3). The
inhibitory effect was sequence-specific as evidenced by the inertness
of ESUP-ARDM. Removal of ATP from the incubation and
stimulation media diminished ~40% of the Ca2+-evoked
release of [3H]NE (32, 34). Note that the ATP-independent
secretion was not affected by ESUP-A. As expected, ESUP-A did not
affect the temperature-sensitive step that follows the
ATP-dependent transition (data not shown). These findings
suggest that ESUP-A interferes with the steps that precede the
ATP-dependent activation of secretory vesicles.
ESUP-A Induces Accumulation of Secretory Granules Near the Plasma Membrane of Chromaffin Cells
The morphology of secretory granules
in chromaffin cells treated with peptides was examined by electron
microscopy (Fig. 4). A main goal was to detect changes
in secretory granule distribution induced by ESUP-A. For comparison
purposes, the effect of BoNT A, known to prevent vesicle docking (35,
36), was investigated. Electron micrographs of permeabilized,
unstimulated chromaffin cells display a random distribution of dense
core secretory vesicles (Fig. 4A). Ca2+
stimulation induces a significant depletion of granules, as evidenced by the reduced number of vesicles in the cytosol (Fig. 4B),
in accord with other reports (33). Notably, the presence of ESUP-A produced an accumulation of vesicles near the plasma membrane (Fig.
4C), similar to that produced by BoNT A (Fig.
4D).
Frequency distribution histograms show that unstimulated (control) chromaffin cells are characterized by the occurrence of two populations of cargo vesicles (Fig. 5A, left): a predominant population with a mean distance to the plasma membrane of ~0.5 µm and a second most frequent with a mean distance of ~2.3 µm. The relative frequency of occurrence of these two vesicle populations was not significantly altered by treating the cells with peptides or BoNT A (Fig. 5, B-D, left). Ca2+-stimulated exocytosis in control cells (Fig. 5A, right) or cells incubated with 100 µM ESUP-ARDM (Fig. 5B, right) decreased the total number of vesicles, as evidenced by the reduction in the area of both granule populations (Figs. 4B and 5A, right). By contrast, treatment with 100 µM ESUP-A or 100 nM BoNT A notably skewed the distribution of vesicles toward the plasma membrane, resulting in the virtual disappearance of the population centered at ~2.3 µm and the ensuing increase of the granule population centered at ~0.5 µm (Fig. 5, C and D, right). The majority of vesicles gathered at the active zone, however, are not in tight contact with the membrane, lending support to the notion that ESUP-A prevents vesicle docking.
A Molecular Mechanism Underlying the Inhibitory Activity of ESUP-AThe main result of our study is that a 20-mer peptide that mimics the C terminus of SNAP-25, effectively inhibits Ca2+-dependent exocytosis in endocrine cells by preventing the docking of cargo vesicles at the plasma membrane. Catecholamine release in permeabilized chromaffin cells exhibits a biphasic time course characterized by an early ATP-independent component, and a slow, ATP-dependent phase (31-35). The ATP-independent phase presumably represents the Ca2+-mediated steps that ultimately lead to fusion, while the ATP-dependent phase may be associated with vesicle priming events (1-7, 33). The final stages of neurosecretion in endocrine cells, therefore, may be described according to the following model (32, 34),
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Given the mechanism depicted in Scheme 1, our results with ESUP-A are
compatible with the concept that the peptide prevents the formation of
the docked state B and the ensuing intermediates in the exocytotic
pathway. The finding that ESUP-A does not affect the fast,
ATP-independent, Ca2+-mediated component (Figs. 1 and 2)
suggests that its site of action is upstream of the fusion event. That
ESUP-A abrogates the slow, ATP-dependent (B C)
component (Fig. 3) is consistent with an inhibition of the formation of
the primed state C, either by blocking the transition from the docked
state B to the first step of the priming process or by inhibiting
vesicle docking. The observation that ESUP-A promotes a massive
accumulation of secretory vesicles near the plasma membrane of
chromaffin cells (Figs. 4 and 5), but not in tight contact, implies
that the peptide blocks the formation of the docked state. This
mechanism of action of ESUP is similar to that proposed for BoNT A
(Table I) (35-37).
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As indicated in Scheme 2 and summarized in Table I, BoNT A and ESUP-A produce comparable effects on the Ca2+-regulated fusion events that lead to transmitter release. Both BoNT A and ESUP-A inhibit the slow component of the Ca2+-dependent exocytosis without affecting the fast component. Both attenuate to a similar extent the ATP-primed exocytosis and not the ATP-independent exocytosis, and both increase the number of vesicles accumulated near the active zone. It appears, therefore, that BoNT A and ESUP-A block exocytosis in chromaffin cells by inhibiting the process of vesicle docking.
The finding that ESUPs are blockers of neurotransmitter release suggests that BoTxs disable the fusion process with such efficacy by a synergistic action of cleaving the substrate molecules that interact through noncovalent interactions to assemble into a fusion complex and of releasing peptide products that block by inhibiting vesicle docking. This implies that saturation with peptides designed to mimic putative sequences that bind to complementary partner sequences on interacting proteins, and combinations thereof, may abrogate vesicle fusion and transmitter release. The notion embodied in the ESUP activity suggests alternative pathways to regulate synaptic vesicle exocytosis and provides novel pharmacological tools to unravel the molecular components and details of the secretory cascade. The fact that these synthetic peptides mimic the action of Clostridial neurotoxins provides clues to develop peptide-based agents that may have practical medical application as potential therapy in disorders associated with involuntary muscle spasms.
We thank B. R. DasGupta for purified BoNT A, M. A. Company for his technical assistance, M. Diez for the drawings, and Dr. E. Fernandez for his expert assistance in morphological vesicle distance distribution analysis.