Truncated SNAP-25 (1–197), Like Botulinum Neurotoxin A, Can Inhibit Insulin Secretion from HIT-T15 Insulinoma Cells

Xiaohang Huang, Michael B. Wheeler, You-hou Kang, Laura Sheu, Gergely L. Lukacs, William S. Trimble and Herbert Y. Gaisano

Departments of Medicine and Physiology University of Toronto (X.H., M.B.W., Y.-h.K., L.S., H.Y.G.) and The Toronto Hospital (H.Y.G., M.B.W.) Toronto, Ontario, Canada M5S 1A8
Divisions of Respirology (G.L.) and Cell Biology (W.S.T.) Hospital for Sick Children Research Institute Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1–197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K+-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the neuroendocrine ß-cell, insulin granule exocytosis appears to be regulated through the actions of a set of proteins, collectively called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) (1), which were originally found in neurons and yeasts (2). The SNARE hypothesis predicts that the cognate vesicle (v-SNARE) and target (t-SNARE) membrane proteins interact to form SNARE complexes that serve to target, dock, and fuse the vesicle to the target membrane (2, 3). Each of these SNARE proteins possess distinct domains that independently bind to their SNARE partners (4, 5, 6). In the cell, the putative v-SNARE on the insulin secretion granule is VAMP-2 (vesicle-associated membrane protein isoform 2) (7, 8), and the putative t-SNAREs on the plasma membrane include SNAP-25 (synaptosomal-associated protein of 25 kDa (8, 9) and several members of the syntaxin family (8, 9, 10).

SNAP-25 proteins possess an amino-terminal domain that binds to syntaxin, a carboxy-terminal that binds to VAMP-2, and a cysteine-rich central domain that attaches this protein to the plasma membrane via palmitoylation (4, 11). Specific cleavage of these SNARE proteins by clostridial neurotoxins blocks neuroexocytosis (12). Botulinum neurotoxin A (BoNT/A) and BoNT/E cleave SNAP-25 at Gln197-Arg198 and Arg180-Ile181, respectively, to remove carboxyl-terminal 9- and 26-amino acid peptides, and this cleavage results in inhibition of neurotransmitter release (13, 14) and insulin secretion (9). In vitro studies have shown that cleavage of SNAP-25 by BoNT/A does not prevent assembly of the SNARE complex, but reduces its stability by inhibiting the acquisition of SDS resistance (5, 6, 15). However, the relationship between these biochemical properties and the observed blockade of secretion in vivo is not clear. Four simple explanations for the actions of BoNT/A are: 1) that the proteolytic action of the toxin depletes the cell of functional SNAP-25 molecules; 2) that the nine-amino acid carboxyl-terminal proteolytic fragment is inhibitory to SNARE complex formation or function; 3) that the 197-amino acid amino-terminal fragment of SNAP-25 forms SDS-sensitive complexes that are nonfunctional, thereby inhibiting exocytosis; or 4) that the cleavage products could interfere with Ca2+ entry into the cell and indirectly block secretion.

The second hypothesis has been supported by experimental evidence. Synthetic peptides of the carboxy-terminal 12 and 20 amino acids of SNAP-25 interfered with Ca2+-evoked secretion in permeabilized neuroendocrine chromaffin cells (16, 17). In the latter report, secretion granules were observed to cluster close to the plasma membrane, suggesting that SNAP-25 plays a role at the docking step in Ca2+-mediated exocytosis. These studies suggested that the mechanism of inhibition by these toxins may be through the generation of the carboxy-terminal peptides released into the cytosol that could then bind to VAMP to block the formation of the SNARE complexes.

In addition, evidence exists in support of the fourth hypothesis, that SNAP-25 binds directly to the plasma membrane Ca2+ channel (18). Coexpression of SNAP-25 and N-type or L-type Ca2+ influx channels in Xenopus oocytes caused an inhibition of inward currents (19). A direct interaction between SNAP-25 and the Ca2+ channel raises the possibility that the BoNT/A cleavage products might inappropriately modulate Ca2+ channel activity in neuronal or neuroendocrine cells.

To gain further insight into the mechanism of BoNT/A actions and SNAP-25 functional domains, we have transfected insulinoma HIT-T15 cells with BoNT/A, full-length SNAP-25, or a SNAP-25 mutant in which the C-terminal nine amino acids had been deleted to mimic the effects of BoNT/A. We found that SNAP-25 proteins lacking the C-terminal nine amino acids were as effective as BoNT/A at inhibiting agonist-evoked insulin secretion from transfected cells, and that this effect was not due to alterations in Ca2+ influx. Transfected cells had significantly higher numbers of intracellular secretory granules, many of which appeared to be in close proximity to the plasma membrane. This suggests that the secretory blockade caused by BoNT/A is caused by the production of the plasma membrane-bound amino-terminal 1–197 fragment of SNAP-25, which acts as an inhibitor of secretion.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of BoNT/A or SNAP-25 (1–197) Mutant in HIT-T15 Cells
To examine the effect of BoNT/A on insulin secretion, HIT-T15 cells were transiently transfected with the cDNA of the light chain of BoNT/A, full-length SNAP-25 (amino acids 1–206), or a SNAP-25 mutant (amino acids 1–197) truncated at the BoNT/A cleavage site in the mammalian expression plasmid pcDNA3. Insulin secretion from HIT-T15 had previously been shown to be inhibited by BoNT/A in permeabilized preparations, but in those studies only modest inhibition was demonstrated (9). The lack of complete inhibition of insulin secretion observed in that study may be due, in part, to inaccessibility of the cleavage site of SNAP-25 proteins that are already complexed to other SNARE proteins (5, 6), temporal limitations of toxin action during leakage of essential soluble factors such as SNAP (20, 21), or the loss of potentially inhibitory peptide cleavage products through permeabilization pores. We chose transfection experiments because they would enable BoNT/A to effectively cleave SNAP-25 before it formed a complex with other SNAREs and would permit the effect to be measured without loss of essential factors or of the cleavage products.

Figure 1Go demonstrates that the transfected proteins are expressed at high levels in HIT-T15 cells. Expression of BoNT/A results in the cleavage of the vast majority of the endogenous SNAP-25 protein (upper panel). This residual intact endogenous SNAP-25 is largely contributed by cells that were either not transfected or expressed BoNT/A poorly. In fact, the transfection efficiency based on the cotransfection with the ß-galactosidase reporter gene ranges from 50–80%. The inability to detect the cleavage product is due, in part, to the lower immunoreactivity of this cleavage product to our antibody, which was generated against the full-length SNAP-25 (8). In support, when exposure was extended (lower panel), the cleavage product appeared (arrowhead). However, the faint appearance of the cleaved fragment suggests the possibility that this fragment might have further undergone accelerated proteolysis. To explore this possibility, we probed the same HIT-T15 lysates expressing BoNT/A with an antibody generated against the N-terminal amino acid 8–29 of SNAP-25 (Transduction Laboratories, Lexington, KY) (data not shown) which showed identical results as those shown in Fig. 1Go. These latter results support the possibility of accelerated proteolytic depletion of SNAP-25 after BoNT/A cleavage.



View larger version (37K):
[in this window]
[in a new window]
 
Figure 1. Immunoreactive SNAP-25 Proteins in Transiently Transfected HIT-T15 Cell Lysates

Upper panel, HIT-T15 cells grown to ~80% confluence on 12-mm wells were transfected transiently with constructed expression vectors for botulinum neurotoxin A-light-chain (BoNT/A), wild-type (WT, SNAP-25, amino acids 1–206), wild-type and BoNT/A (WT + BoNT/A), or the SNAP-25 (amino acids 1–197) mutant, compared with control untransfected cells. Lower panel, the same blot in the lower panel for the BoNT/A transfection but exposure was 5-fold longer to visualize the cleaved protein (arrowhead). Ten micrograms of protein of each sample were loaded, and blots were probed with anti-SNAP-25 antisera. This is a representative of four independent experiments.

 
Figure 1Go (upper panel) also shows that transfection with the full-length SNAP-25 protein results in SNAP-25 levels significantly higher than endogenous levels. Cotransfection with the full-length SNAP-25 and BoNT/A resulted in virtually complete cleavage of the expressed SNAP-25, as evidenced by the increased mobility of the immunoreactive species. Complete cleavage of the cotransfected product is not surprising since transfected cells typically coexpress both plasmids (as in Fig. 2Go). The antibody used in these studies was able to detect both full-length and truncated proteins. Transfection of the SNAP-25 (1–197) mutant resulted in equivalently high levels of expression compared with the full-length protein, and the mobility of the product was identical to that resulting from BoNT/A cleavage.



View larger version (59K):
[in this window]
[in a new window]
 
Figure 2. Cellular Location of SNAP-25 Proteins of Transiently Transfected HIT-T15 Cells Labeled with anti-SNAP-25 Antibody and Detected by Laser Scanning Confocal Microscopy

Untransfected HIT-T15 cells plated on coverslips were probed with anti-SNAP-25 antibodies (a) alone or (b) preincubated with recombinant GST-SNAP-25 proteins which completely blocked the SNAP-25 signal. HIT-T15 cells were transfected with constructed vectors for (c) wild-type SNAP-25 (amino acids 1–206) or (e) BoNT/A and probed with the anti-SNAP-25 antibody. Coexpression with the GFP (panels d and f, indicated by arrows) shows the corresponding increased SNAP-25 signal in cells overexpressing the wild-type SNAP-25 protein (in c) and corresponding decreased SNAP-25 signal in cells expressing the BoNT/A toxin (in panel e). Panels c-f demonstrate that the transfected cells (indicated by arrows) were able to take up both plasmids. Cells transfected with (g) wild-type SNAP-25 + BoNT/A or (h) SNAP-25 mutant (amino acids 1–197) and were also probed with the anti-SNAP-25 antibody, and the arrows point to the transfected cells overexpressing the SNAP-25 proteins. Two days after transfection, the coverslips were probed with rabbit anti-SNAP-25 antibody, fixed and labeled with secondary antibodies, and then visualized on a laser scanning confocal microscope as described in Materials and Methods. Bar, 10 µm.

 
To determine the localization of transfected SNAP-25 proteins and the effect of BoNT/A expression on that localization, laser confocal immunofluorescence microscopy was performed using antibodies specific to SNAP-25 and coexpression of the green fluorescence protein (GFP). In untransfected control HIT-T15 cells, SNAP-25 was found on the plasma membrane consistent with its role as a plasma membrane t-SNARE for insulin granule exocytosis (Fig. 2AGo). Specificity of this antibody staining was demonstrated by the complete blockade of this signal when the antibody was preincubated with recombinant GST-SNAP-25 protein (Fig. 2BGo). Coexpression of the wild-type SNAP-25 (amino acids 1–206) protein along with the GFP shown in Fig. 2Go, C and D, confirmed that the transfected cells (indicated by arrows) expressed both plasmids. In all experiments performed throughout these studies, transformation frequency was quantitated, either as a measure of the number of cells overexpressing the SNAP-25 proteins, those expressing cotransfected GFP, or cotransfection of ß-galactosidase in control wells, to be between 50% and 80%. The intensity of the SNAP-25 signal was not altered when the GFP alone was expressed (data not shown). Transient transfection with BoNT/A along with the GFP in Fig. 2Go, E and F, demonstrated a diminished intensity in the SNAP-25 signal in the transfected cells (indicated by arrows), which corresponds to the profound proteolysis of SNAP-25 as measured by Western blot (Fig. 1Go). However, there was no effect on the plasma membrane localization of the cleaved SNAP-25 protein (Fig. 2EGo). Similarly, transfection of both BoNT/A and the wild-type SNAP-25 (1–206) in Fig. 2GGo shows that the cleavage of the overexpressed SNAP-25 proteins by BoNT/A (indicated by arrows) did not alter their plasma membrane localization. This result was identical to that obtained in Fig. 2HGo with cells transfected with the SNAP-25 (1–197) mutant protein (indicated by arrows), which also did not result in mislocalization (Fig. 2GGo). In Fig. 2Go, C–G, cells that were not transfected showed a similar low intensity of plasma membrane staining equivalent to the control untransfected cells (Fig. 2AGo), whereas cells that were inefficiently transfected showed intermediate signal intensity. These results, taken together, indicate that overexpression of the wild-type or mutant form of SNAP-25, or of BoNT/A, did not affect the fidelity of targeting of these SNAP-25 proteins to their normal destination, the plasma membrane. These modified SNAP-25 proteins should therefore be able to interact with plasma membrane proteins, including their SNARE partners and the L-type Ca2+ channel, located in the plasma membrane.

Effects of BoNT/A or SNAP-25 (1–197) Mutant Transfections on Potassium- or Glucose-Stimulated Insulin Secretion
K+ depolarization of the endocrine cell plasma membrane activates the distal secretory machinery by directly inducing Ca2+ influx, which in turn evokes insulin exocytosis (22). To examine the effect of the treatments used above on evoked insulin secretion, we treated control and transfected HIT-T15 cells with Ca2+-containing high K+ (30 mM) buffer or a control buffer with low K+ (4.8 mM) in a manner similar to that described by Boyd et al. (23). Figure 3Go shows the insulin secretion evoked by the high K+ buffer above basal insulin release caused by low K+ buffer. In control untransfected cells, the high K+ buffer evoked insulin release of 2- to 4-fold that released by the low K+ buffer (data not shown). To minimize inter- and intraassay variations and possible variation in transfection efficiency, the following controls were performed. As a control for the effect of transfection on release, cells transfected with the empty vector had no effect on insulin release compared with untransfected cells (data not shown). Cells in each set of experiments were from a single plate of cells of the same passage, plated in identical manner into each well. Triplicate wells for each variable and control were performed for each experiment, and at least three independent experiments were performed. The viability and confluence of the cells in each well were confirmed to be uniform by visual examination. Transfection efficiencies in the range of 50–80% were confirmed by coexpression of the ß-galactosidase gene in a control well performed in every set of experiments.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 3. Potassium-Stimulated Insulin Release from HIT-T15 Cells

HIT-T15 cells were transfected as described in Materials and Methods with BoNT/A in panel A, wild-type (WT, SNAP-25 amino acids 1–206) + BoNT/A in panel B, or SNAP-25 (1–197) mutant in panel C. Each experiment was performed and compared with control untransfected cells. In panel B, cells transfected with the wild-type SNAP-25 protein alone were also compared. The release of insulin from the cells during a 1-h incubation period with 30 mM K+ or 4.8 mM K+ (basal level) was determined by RIA as described in Materials and Methods. Each value shown is the means ± SEM of the insulin released above basal levels. The number of wells for each value and their statistical significance are indicated in Results.

 
Figure 3AGo demonstrates that transient expression of BoNT/A reduced the high K+-evoked insulin release to 0.796 ± 0.203 ng/well compared with 1.984 ± 0.261 ng/well for untransfected cells (n = 9 wells, 3 experiments, P = 0.001), an approximately 60% reduction. Figure 3BGo demonstrates that cotransfection of BoNT/A, along with the wild-type native SNAP-25 protein, resulted in a high K+-evoked insulin release of 1.005 ± 0.18 ng/well (n = 13 wells, 4 experiments) which was a significant reduction of ~65% and ~62%, respectively, compared with the insulin released from control (2.905 ± 0.458 ng/well, P = 0.002) or wild-type SNAP-25 transfected cells (2.656 ± 0.63 ng/well, P = 0.002).

It is possible that BoNT/A inhibition of insulin release may be mediated by its proteolytic effects on undefined substrates other than SNAP-25. We have therefore examined the effects on insulin secretion resulting from the transfection of a SNAP-25 mutant (amino acids 1–197) in which the terminal nine amino acids corresponding to the BoNT/A cleavage site are truncated (Fig. 3CGo). This strategy further allows us to isolate the distinct effects of amino acids 1–197 domain of SNAP-25 independent of the C-terminal nine-amino acid fragment resulting from BoNT/A proteolytic cleavage. Insulin release evoked by the high K+ was 1.007 ± 0.23 ng/well (n = 12 wells, 4 experiments) for the SNAP-25 mutant and 3.09 ± 0.76 ng/well for the control wells, which was a significant reduction of ~67% (P = 0.006).

These studies were also conducted using a physiological secretagogue, glucose, at a concentration known to maximally stimulate insulin secretion (24, 25). As in Fig. 3Go, cells were transfected with BoNT/A (Fig. 4AGo) or the SNAP-25 (1–197) mutant protein (Fig. 4BGo) compared with control cells transfected with the empty vector. To evoke secretion, cells were incubated in Krebs-Ringer bicarborate (KRB) media containing 10 mM glucose (1 h, 37 C), which released insulin that was generally 2-fold of basal insulin secretion. Figure 4Go shows evoked insulin release that was subtracted from basal insulin release at 0 glucose concentration. We found that transfection of BoNT/A reduced insulin release from the control value of 1.70 ± 0.09 ng/well to 0.83 ± 0.08 ng/well (n = 15 wells, 4 experiments, P = 0.0002), or a reduction of ~51% (Fig. 4AGo). Overexpression of the SNAP-25 (1–197) mutant (Fig. 4BGo) caused a reduction of insulin release from a control value of 1.53 ± 0.49 ng/well to 0.26 ± 0.06 ng/well (n = 9 wells, 3 experiments, P = 0.027), or a reduction of ~83%. The more marked inhibition observed with these latter studies is due largely to the higher transfection rate in this set of experiments. These results, demonstrating similar inhibition of acute glucose-evoked insulin release by transient expression of either BoNT/A or of the SNAP-25 (1–197) mutant, are consistent with the results obtained with acute high K+-evoked insulin release in Fig. 3Go.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 4. Glucose Stimulation of Insulin Release from HIT-T15 Cells

HIT-T15 cells were grown and transiently transfected with constructed vectors of (A) BoNT/A or (B) SNAP-25 (amino acids 1–197) mutant as described in Fig. 3Go. Control cells were transfected with the vector only. The release of insulin from the cells during a 1-h incubation period with no glucose (basal levels) or 10 mM glucose was determined by RIA as described in Materials and Methods. Each value shown is the means ± SEM of the insulin released above basal levels. The number of wells for each value and their statistical significance are indicated in Results.

 
The residual insulin secretion of ~20–40% observed with the wells transfected with either the BoNT/A (Fig. 3Go, A and B) or SNAP-25 (1–197) mutant (Fig. 3CGo) genes is likely to be due to cells that were either not transfected or that express lower levels of cleaved or mutant SNAP-25 proteins. Nonetheless, the levels of reduction in evoked insulin secretion of ~50–80% in these studies are remarkably comparable to the efficiency of transfection of 50–80% based on the ß-galactosidase assay and ~67% based on confocal microscopy studies (mean of 17 randomly chosen micrographs as in Fig. 2Go). Taken together, these studies strongly suggest that high K+- or glucose-evoked insulin secretion from the transfected cells were markedly, if not totally, inhibited. Furthermore, the uniform inhibition of evoked insulin secretion from either the BoNT/A- or SNAP-25 (1–197) mutant-transfected cells demonstrate that expression of the SNAP-25 (1–197) mutant proteins could account for the entire BoNT/A-mediated inhibition of evoked insulin release.

We also compared the basal insulin release in the studies performed in Figs. 3Go and 4Go and found no statistical difference between the cells expressing BoNT/A or truncated SNAP-25 mutant and control cells (data not shown), suggesting no effects of these treatments on constitutive insulin exocytosis.

Effects of BoNT/A or SNAP-25 (1–197) Mutant Transfections on Potassium-Mediated Plasma Membrane Depolarization-Evoked Ca2+ Influx
Because of the recent reports that SNAP-25 could directly modulate Ca2+ channel kinetics, including the neuroendocrine L-type channel (19), it is possible that these transfections could affect Ca2+ influx via this or other Ca2+ influx channels to inhibit the insulin secretion, as observed in Figs. 3Go and 4Go. We have therefore performed fluorescent video-imaging of transfected HIT-T15 cells loaded with the Ca2+ indicator dye fura 2AM and perfused with buffers containing different concentrations of Ca2+ and K+ (Fig. 5Go). The HIT-T15 cells studied were transfected with the empty vector (control, Fig. 5AGo), BoNT/A (Fig. 5BGo), or the SNAP-25 (1–197) mutant protein (Fig. 5CGo). Transfection efficiency was again confirmed by coexpression of GFP performed as a control in each experiment. Fluorescent ratio intensity values at 340 nm and 380 nm were obtained for each of the cells visualized in the field, and the data are expressed as the ratio of 340 nm to 380 nm (Fig. 5Go). There was no detectable difference in the ratio values between the cells within each coverslip during exposure to low K+ or high K+ buffers. We therefore randomly chose a field within each coverslip, and the ratio values obtained from all of the cells within the field were analyzed together as described in Materials and Methods. Basal Ca2+ levels were first determined with a low K+, 1 mM Ca2+ buffer, and the ratio values (mean ± SEM) obtained for the control (0.75 ± 0.01), BoNT/A (0.75 ± 0.01), and SNAP-25 (1–197) mutant (0.73 ± 0.01) transfected cells as indicated in Table 1Go were not significantly different. To better assess Ca2+ influx activity, ratio values (mean ± SEM) were obtained at 0.5 min and 3 min after stimulation (indicated by the first arrows) with the high K+, 1 mM Ca2+ buffer. As shown in Table 1Go, ratio values at each of these time points were not significantly different. Upon removal of the extracellular Ca2+ (in 10 mM EGTA, indicated by the second arrows), the intracellular Ca concentration ([Ca2+ ]i) uniformly and abruptly returns to basal levels. This latter result, together with a control experiment using a high K+, nominal free Ca2+ (no added Ca2+ in 10 mM EGTA) buffer that gave no rise in [Ca2+]i (not shown), indicate that the rise in [Ca2+]i evoked by the high K+ buffer was generated entirely from an extracellular source coming through voltage-dependent Ca2+ influx channels. These results, taken together, indicate that the inhibition of insulin secretion caused by these transfections does not seem to involve a direct effect on the Ca2+ influx into the ß-cell.



View larger version (11K):
[in this window]
[in a new window]
 
Figure 5. Effects on Ca2+ Influx Evoked by Potassium-Mediated Plasma Depolarization in Single HIT-T15 Cells

HIT-T15 cells were plated on coverslips and transfected with the empty vector (Control) in panel A, BoNT/A in panel B, or SNAP-25 (amino acids 1–197) mutant in panel C as described in Materials and Methods. After loading, the cells were with fura-2AM, the coverslip was mounted onto a perfusion chamber and onto the microscope, and a field of cells was randomly chosen. Fluorescent images of cytosolic Ca2+ levels of single cells were then obtained as described in Materials and Methods. The coverslips were first perfused with a low K+ (4.8 mM), 1 mM Ca2+ buffer until a steady baseline was obtained. The coverslip was then perfused with a high K+ (30 mM), 1 mM Ca2+ buffer as indicated by the first arrows, followed by removal of the Ca2+ (30 mM K+, 0 mM Ca2+) as indicated by the second arrows. Each value is expressed as mean ± SEM of the mean values of each field of cells (15–25 cells per field) randomly picked from a coverslip, and at least seven coverslips from two to three independent experiments were analyzed as also described in Materials and Methods. The Ca2+ values were not significantly different (see Table 1Go).

 

View this table:
[in this window]
[in a new window]
 
Table 1. Basal and Potassium-Evoked Rise in Cytosolic Calcium Levels in Transiently Transfected HIT-T15 Cells

 
BoNT/A or SNAP-25 (1–197) Mutant Transfections Resulted in Accumulation of Insulin Granule Near the Plasma Membrane
Another possible explanation for the decreased insulin release from cells expressing either BoNT/A or SNAP-25(1–197) could be that the transfected cells simply produced less insulin. To examine the quantity and subcellular location of insulin-containing granules, we performed electron microscopy of these cells (Fig. 6Go). Since not all cells were transfected in any treatment, experiments were performed in double blind conditions, and 23 micrographs were randomly taken from three independent experiments for each vector. Parallel control transfections were performed to confirm that the transfection efficiency was more than 50%. Transfection with the ß-galactosidase reporter gene did not affect the number of granules compared with control untransfected cells (not shown). In all cases, the number of insulin granules in the HIT-T15 insulinoma cell line was relatively sparse compared with normal islet ß-cells (our unpublished observation). To accurately determine the cellular distribution of the accumulated granules in these transfected cells, we performed morphometric analysis of these randomly chosen micrographs as a function of the distance from the plasma membrane to the nucleus, and data were analyzed and expressed as frequency distribution histograms of the number of granules per µm2 of cytosolic space (Fig. 6Go). Only 16 of the 23 micrographs for each of the vector-contained cells with the appropriate distance of at least 3.6 µm of cytosolic space between the plasma membrane and nuclei were analyzed. In the control cells, the insulin granules were randomly distributed at a density of about 1 granule/µm2 from the nucleus to 0.45 µm from the plasma membrane, but within 0.45 µm of the plasma membrane that density approximately doubled. The granule distribution immediately adjacent to the nucleus in BoNT/A and SNAP-25 (1–197) mutant transfected cells appeared similar to the control cells. However, in contrast to control cells, the granule densities in BoNT/A and SNAP-25 mutant transfected cells rise progressively and uniformly as the plasma membrane was approached, and were highest at within 0.45 µm from the plasma membrane, where they were 2-fold higher than in control cells. These results demonstrate that the long-term effects of the BoNT/A or truncated SNAP-25 (amino acid 1–197) expression caused an accumulation of insulin secretion granules. This pattern of accumulation of the insulin granules suggests not only an exocytotic blockade at the plasma membrane, but might also affect additional more proximal steps in the mobilization of the insulin secretion granules. Alternatively, the blockade of exocytosis might cause some feedback in the granule translocation process.



View larger version (14K):
[in this window]
[in a new window]
 
Figure 6. Subcellular Distribution of Insulin Granules in HIT-T15 Cells

HIT-T15 cells were grown in RPMI 1640 and transiently transfected with either the empty vector for control (in panel A) or the constructed vectors of BoNT/A (in panel B) or SNAP-25 (amino acids 1–197) mutant (in panel C) as described in Figs. 3Go and 4Go. The cells were then processed for ultrastructural study as described in Materials and Methods. The micrographs obtained at a uniform original magnification of 6,600x for each vector were subjected to morphometric analysis using the NIH Image analysis program. This figure shows the frequency distribution histograms of the number of insulin granules as a function of the distance from the plasma membrane (0 µm) to the nucleus (3.6 µm). The surface area within each 0.45 µm bin width from the plasma membrane was determined and the number of insulin granules within the bin counted. The data are then expressed as the number of granules per µm2 of cytosolic surface area of each 0.45 µm bin width. Of the 23 random micrographs obtained from three independent experiments for each vector, only 16 micrographs per vector contained cells with the appropriate cytosolic space between the plasma membrane and the nucleus for analysis. Each bin value is expressed as the mean ± SEM (n = 16 micrographs).

 
It is conceivable that the long-term effects of BoNT/A or truncated SNAP-25 expression might affect processes other than exocytosis of insulin secretion granules, including insulin synthesis and insulin vesicular loading. We have therefore determined the total insulin content of these cells (Fig. 7Go) and compared the results to the total number of insulin granules in these cells. The total insulin content cells expressing BoNT/A and truncated SNAP-25 mutant were 236.84 ± 19.63 ng/well and 244.64 ± 17.36 ng/well, respectively, compared with 188.75 ± 11.99 ng/well in the control cells (n = 15 wells, 5 experiments). This was a small but significant increase of only 25% for BoNT/A (P = 0.004) and 30% for truncated SNAP-25 mutant (P = 0.0004) transfected cells compared with control cells. We then counted the total number of granules from each bin in Fig. 6Go as a function of the total cytosolic surface occupied by these granules. There were 1.03 ± 0.13 granules/µm2 (n = 16 micrographs) for the control cells, which was significantly different from BoNT/A (1.81 ± 0.18 granules/µm2, n = 16 micrographs, P = 0.001) and SNAP-25 (1–197) mutant (1.86 ± 0.23 granules/µm2, n = 16 micrographs, P = 0.004) transfected cells. The granule density and total insulin content between BoNT/A and SNAP-25 (1–197) mutant transfected cells were similar. This granule density ratio of 2:1 of either BoNT/A or SNAP-25 (1–197) mutant transfected cells compared with the control cells was preserved when the rest of the 23 micrographs per vector were accounted for (data not shown). The incremental increase in insulin granule density is about 3-fold that of the incremental increase in total insulin content in these cells. Taken together, these results suggest that the small increase in total insulin content effected by the long-term treatments with the BoNT/A or SNAP-25 mutant are likely to be contained mostly, if not entirely, in the insulin secretion granule compartment. Furthermore, these treatments are unlikely to have caused a significant effect on insulin synthesis or nascent insulin content in other cellular compartments.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 7. Total Insulin Content in Transiently Transfected HIT-T15 Cells

HIT-T15 cells were treated and transiently transfected with either the empty vector for control (in panel A) or the constructed vectors of BoNT/A (in panel B) or SNAP-25 (amino acids 1–197) mutant (in panel C) as in Fig. 6Go. The cells were then harvested in 1 M acetic acid, lysed by two freeze-thaw cycles, and appropriately diluted, and insulin was determined by RIA.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin exocytosis in the islet cell shares many attributes with neurotransmitter release including the presence of neuronal SNAREs VAMP-2, SNAP-25, and syntaxin 1 in islets and insulinoma cells, each of which have all been shown to participate in regulating insulin secretion (7, 8, 9, 10, 26). Although the use of Clostridial neurotoxins has been instrumental in gaining insights into the functions of these SNARE proteins in the cell, the precise mechanism by which these toxins block secretion had not been clear. We have therefore attempted to examine the molecular basis for BoNT/A actions in the cell by transient expression of BoNT/A or the SNAP-25 (1–197) mutant in which the C-terminal nine amino acids are deleted.

Our work has demonstrated that transient transfection of the truncated version of the SNAP-25 protein (amino acids 1–197) identical to the larger fragment produced by BoNT/A cleavage results in a major inhibition of insulin secretion, which is at least equivalent to that caused by transfection of BoNT/A itself. Therefore, the inhibition of both K+- and glucose-stimulated insulin secretion by BoNT/A may be due, in part, to the accumulation of the N-terminal amino acid 1–197 fragment of SNAP-25, which remains bound to the plasma membrane. This does not preclude a role of accelerated degradation of the BoNT/A cleaved N-terminal amino acid 1–197 fragment of SNAP-25 as shown in Fig. 1Go, or a role for the smaller C-terminal nine-amino acid cleavage product release into the cytosol (17), but indicates that these are not solely responsible for the BoNT/A inhibition. These three mechanisms could cooperate in vivo to cause inhibition of insulin release.

The remarkable similarity between the degree of inhibition of insulin secretion and the frequency of transfection suggests that within the individual transfected cells, secretion may have been completely inhibited. This inhibition of insulin release is considerably greater than that observed with BoNT/A by Sadoul et al. (9) using a permeabilized assay evoked by Ca2+ stimulation, but similar to that reported by Boyd et al. (23) using the electroporation technique. The likely explanation for these differences is that electroporation causes only a transient permeabilization of the cells, thereby avoiding a rundown of cytosolic proteins, and, in addition, allows a more prolonged and effective action of the internalized neurotoxin. Taken together, these results demonstrate that the N-terminal 1–197 domain of SNAP-25 acting at its native t-SNARE location on the plasma membrane is capable of inhibiting insulin secretion.

Several lines of evidence suggest that components of the SNARE complex may interact directly with the Ca2+ channels. Bokvist et al. (27), using the patch clamp technique, suggested that the insulin granules and the L-type channel are in close proximity, if not colocalized, in the ß-cell. This is analogous to the situation in the neuron where the SNARE core complex has been shown to directly interact with the neuronal N-type channel (18). Furthermore, Wiser et al. (19) found coexpression of either N-type or L-type Ca2+ channels with SNAP-25 in Xenopus oocytes resulted in inhibition of inward Ca2+ current. Thus, the proximity between SNAP-25 and the L-type channel raises the possibility that the products of BoNT/A action may disrupt Ca2+ channel activation. However, our microfluorimetry studies demonstrated that basal cytosolic Ca2+ levels, as well as K+-evoked Ca2+ influx determined over at least 3 min of continued perfusion, were identical between cells expressing either BoNT/A or SNAP-25 (1–197) mutant constructs, and control HIT-T15 cells. Taken together, these studies indicate that the inhibitory effects on insulin secretion by the modified SNAP-25 proteins are likely to be independent of the ß-cell Ca2+ influx channels.

Thus, the expression of either BoNT/A or the truncated SNAP-25 (1–197) cleavage product may lead to the same result: the accumulation of products that compete with the endogenous SNAP-25 for binding to syntaxin and VAMP during the processes of docking and/or fusion. Syntaxin binds to a region within the N-terminal 100 amino acids of SNAP-25, whereas the C terminus of SNAP-25 binds to VAMP-2 (5, 6, 15). In vitro studies have demonstrated that either a BoNT/A-cleaved SNAP-25 protein or a recombinant truncated SNAP-25 protein corresponding to our SNAP-25 (amino acids 1–197) mutant weakened the binding of these truncated SNAP-25 proteins to VAMP-2 compared with the native SNAP-25 but did not affect their binding to syntaxin 1. These truncated SNAP-25 proteins therefore remained able to assemble into a ternary complex with VAMP-2 and syntaxin 1, but this complex had a reduced ability to assemble in an SDS-resistant form. The SDS-resistant property of the SNARE complex may reflect a low-energy state for the protein complex required for subsequent functional activation by the action of {alpha}-soluble N-ethylmaleimide-sensitive attachment protein ({alpha}SNAP) and N-ethylmaleimide-sensitive factor (NSF). Biophysical studies are now required to determine how the loss of the C-terminal nine amino acids of SNAP-25 can alter its function so profoundly.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The HIT cell subclone T15 was a gift from Dr. P. Robertson (University of Minnesota, Duluth, MN). Plasmid cDNAs for full-length BoNT/A-light chain and SNAP-25 were generous gifts from H. Niemann (Hanover, Germany) and M. Wilson (La Jolla, CA), respectively.

Generation of Expression Construct and Transfection of Cells
For the SNAP-25 mutant, a sense oligonucleotide primer (5'-ACTACCATGGCCGAGGACGCAGACATG-3') and antisense primer (5'-CTATTGGTTGGCTTCATCAATTCTGGT-3') were generated according to the published sequence. The PCR products were cloned into PCRII vector (Invitrogen, San Diego, CA) and then subcloned into a pcDNA3 vector. The identity of the PCR products of the predicted size was confirmed by DNA sequencing using T7 polymerase (Pharmacia, Piscataway, NJ). Plasmids containing the corresponding full length SNAP-25 wild-type (1–206) and Botulinum A-light-chain cDNAs were subcloned into pcDNA3 as with the SNAP25 mutant (amino acids 1–197). The HIT-T15 cells were grown at 37 C in 5% CO2/95% air in RPMI 1640 medium supplemented with 20 mM glutamine, 10% FCS (GIBCO, Gaithersburg, MD), penicillin (100 U/ml), and streptomycin (100 mg/ml). After the cells were grown to a density of 3 x 106 cells per well in 12-well (20 mm) plates, these cells were transiently transfected with 2 µg plasmid DNA in Optimum medium the following day using Pfx-1 lipid (Perfect Lipid, Invitrogen) at a DNA/lipid ratio of 1:6 (wt/wt). After a 4-h incubation at 37 C in 5% CO2, normal RPMI 1640 medium was added. The medium was discarded the following day and replaced with fresh RPMI 1640 medium. Transfection efficiency was determined by using pcDNA3/His/Lac Z (Invitrogen) as a control plasmid for transfections followed by determination of ß-galactosidase expression, and by counting the cells that overexpressed the transfected proteins in the confocal microscopy studies.

Immunoblotting
Affinity-purified rabbit antisera generated against the recombinant full-length SNAP-25 was a generous gift from M. Bennett (University of California, Berkeley, CA). This antibody has been previously reported and validated to be specific for SNAP-25 (8, 28). SDS-PAGE and immunoblotting were performed as previously described (8). Protein concentrations of each cell lysate sample were initially determined by the Bio-Rad (Richmond, CA) protein assay kit. The cell lysates were then dissolved in sample buffer and the proteins separated on a 15% polyacrylamide gel. These proteins were then transferred to a nitrocellulose membrane, and the blots were then incubated with the primary antibody (anti-SNAP 25 antibody, 1:1000 dilution in 1% BSA, 0.05% Tween-20 in PBS) for 1.5–2 h at room temperature. Immunodetection was by enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL).

Insulin Secretion
Twenty four hours after transfection, the media were changed and then further incubated for an additional 24 h. These cells were cultured to ~80% confluence before stimulation.

Potassium (K+) and Glucose Stimulation
K+ stimulation was conducted according to a slightly modified method described by Boyd et al. (23). The transfected or control cells were washed and then incubated in 0.5 ml low K+-KRB buffer containing 4.8 mM KCl, 129 mM NaCl, 5 mM NaHCO3, 1.2 mM KH2PO4, 1 mM CaCl2, 1.2 mM MgSO4, 0.5 mM glucose, and 10 mM HEPES, pH 7.4, for 0.5 h. The media were then discarded and then exchanged with 0.5 ml of the same buffer containing either low K+ (4.8 mM KCl, for basal levels) or high K+ in which the KCl concentration was increased to 30 mM and NaCl was reduced to 104 mM for 1 h at 37 C. For glucose stimulation, the wells were exposed to the KRB buffer (with 4.8 mM KCl) in which glucose was either removed (for basal levels) or increased to 10 mM, and incubation was carried out for 1 h at 37 C. At the end of incubation, the media were collected and then centrifuged at 3000 x g for 3 min to remove detached cells, and the resulting upper half of the supernatants were collected. Both supernatants and some the cells (collected by scraping the plates) were frozen at -20 C. Insulin in the samples was then assayed within 1 week using a rat insulin RIA kit (Linco, St. Charles, MO) according to manufacturer’s instructions. Appropriate dilutions of the samples were carried out to allow the values to fall into the standard curve.

Because of the inherent variability of transfection efficiency and the effects of the passage of the cell lines, cells from all wells in each experiment performed were always from the same plate of cells. Every experiment always included all of the variables indicated in each figure being compared, and at least three to five independent experiments with triplicate or four replicate wells per experimental variable were performed. Furthermore, the protein concentrations of each well, determined to be 392 ± 10.4 (n = 8 independent experiments), and confluence of cells in each well (~80%) were nearly identical within and between experiments. Values shown in the figures are the mean increase above basal levels, which were also determined in triplicate wells for each experiment. The data are expressed as means ± SEM of the total number of wells and analyzed by Student’s t test. A P value <0.05 indicates that the difference is significant.

Confocal Immunofluorescence Microscopy
Microscopy was performed as previously described (8, 28). Briefly, the cells were plated on round polylysine-coated coverslips and placed in 12-well plates. Forty eight hours after transfection, the cells were fixed in 2% formaldehyde for 0.5 h at room temperature, blocked with 5% normal goat serum with 0.1% saponin for 0.5 h at room temperature, and then immunolabeled with primary antibodies (1:100 anti-SNAP-25 antibody) overnight at 4 C. After rinsing with 0.1% saponin/PBS, the coverslips were then incubated with the appropriate rhodamine-labeled secondary antiserum for 1 h at room temperature and then mounted on slides in a fading retarder, 0.1% p-phenylenediamine in glycerol, and examined using a laser scanning confocal imaging system (Zeiss, Oberkochen, Germany). Transfected cells were identified by coexpression and visualization of the GFP (Clontech, Palo Alto, CA).

Single-Cell [Ca2+]i Determinations
Cells plated on polylysine-coated coverslips were loaded with 0.5 µM fura-2 AM in serum-free medium for 20 min at 37 C, washed, and then further incubated in dye-free medium for an additional 10 min at 37 C. The coverslip was then mounted on a chamber and perfused continuously at 25 C with the test media, which uniformly contain 5 mM NaHCO3, 1.2 mM MgSO4, 2.8 mM glucose, 10 mM HEPES, pH 7.4, but with variable concentrations of K+/Na+ [4.8 mM KCl with 129 mM NaCl for low K+ buffer, and 30 mM KCl/103.8 mM NaCl for high K+ buffer] and Ca2+ [0 or 1 mM CaCl2]. Quantitative fluorescence imaging of single cell [Ca2+]i at low light levels was then performed in a manner similar to what we had previously described for pancreatic acinar cells (29) using an Axiovert 100 inverted microscope (Zeiss) coupled to a cooled CCD camera (Princeton Instruments Inc., Princeton, NJ). Fluorescent images (excitation, 340 and 380 nm; emission, 510 nm) were acquired every 20–30 sec, and ratios of fluorescence intensities were calculated for each cell (15–20 cells per field per coverslip) after subtracting the background fluorescence using Metafluor software (Universal Imaging Corp., West Chester, PA). For each condition, two to three independent experiments with three to four separate coverslips per experiment or a total of at least seven coverslips per condition were performed. The fluorescent ratio values of the cells in each coverslip (15–25 cells per field) were integrated, and the mean of these values was determined at basal, 0.5 min, and 3 min after stimulation with the high K+ medium. The final data expressed are the mean ± SEM of the mean values obtained from each coverslip. Transfection efficiency for each experiment was confirmed to be ~50–80% by coexpression and visualization of either the GFP or ß-galactosidase protein in a control coverslip.


    ACKNOWLEDGMENTS
 
Electron Microscopy

Forty eight hours after transfection, both transfected cells and control cells (untransfected or transfected with pcDNA3/His/Lac Z) were fixed with 0.5% glutaraldehyde/4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 20 min at room temperature, followed by osmication with 1% aqueous OsO4 for 30 min. The samples were then dehydrated according to standard procedures and embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were stained with uranyl acetate for 5 min and then treated with lead citrate for 5 min and observed with a H600 transmission electron microscope (Hitachi, Tokyo, Japan) at 80 kV at a uniform magnification of 6600x. Twenty three micrographs were randomly taken for each condition from three independent experiments by a morphology technician not involved with our laboratory. To determine the granule density distribution within each cell, a method similar to that described by Gutierrez et al. (17) in chromaffin cells using the NIH Image analysis program and Adobe Photoshop (Adobe Systems Inc., San Jose, CA) software were used. Insulin granules, identified as a dense core granule with a distinct membrane, were counted within a bin width of 0.45 µm from the plasma membrane to the nucleus, with the plasma membrane at 0 µm and the nuclear membrane at 3.6 µm. In our study, we further determined the surface area within each of the 0.45 µm bin width of cytosolic space using the same NIH Image program, and the number of granules was expressed as a function of this cytosolic surface area. A secretory granule density distribution histogram was then constructed from these data in Fig. 6Go. Significance was measured by Student’s t test.


    FOOTNOTES
 
Address requests for reprints to: Herbert Y. Gaisano, Room 7226 Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: herbert.gaisano{at}utoronto.ca

Supported by grants from Canadian Diabetes Association, Juvenile Diabetes Foundation, Eli Lilly-Banting and Best Diabetes Research Program at the University of Toronto (to H.Y.G. and M.B.W.) and the Medical Research Council of Canada (MT-13169 to H.Y.G. and W.S.T.). H. Gaisano is a recipient of the American Gastroenterology Association/Industry (Pharmacia and Upjohn) Research Scholar Award.

Received for publication September 30, 1997. Revision received January 22, 1997. Accepted for publication March 9, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Wollheim CB, Lang J, Regazzi R 1996 The exocytotic process of insulin secretion and its regulation by Ca2+ and G-proteins. Diabetes Rev 4:276–297
  2. Sudhof TC 1995 The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375:645–653[CrossRef][Medline]
  3. Rothman JE, Warren G 1994 Implications of the SNARE hypothesis for intracellular membrane topology and dynamics. Curr Biol 4:220–233[Medline]
  4. Chapman ER, An S, Barton N, Jahn R 1994 SNAP-25 a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils. J Biol Chem 269:27427–27432[Abstract/Free Full Text]
  5. Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Sudhof TC 1994 Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J 13:5051–5061[Abstract]
  6. Pelligrini L, O’Connor VO, Lottspeich F, Betz, H 1995 Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion. EMBO J 14:4705–4713[Abstract]
  7. Regazzi R, Wollheim CB, Lang J, Theler J-M, Rossetto O, Montecucco C, Sadoul K, Weller U, Plamer M, Thorens B 1995 VAMP-2 and cellubrevin are expressed in pancreatic ß cells and are essential for calcium but not for GTP{gamma}S-induced insulin secretion. EMBO J 12:2773- 2730[Abstract]
  8. Wheeler MB, Sheu L, Ghai M, Bouquillon A, Grondin G, Weller U, Beaudoin AR, Bennett MK, Trimble WS, Gaisano HY 1996 Characterization of SNARE protein expression in ß cell lines and pancreatic islets. Endocrinology 137:1340–1348[Abstract]
  9. Sadoul S, Lang J, Montecucco C, Weller U, Regazzi R, Catsicas S, Wollheim CB, Halban PA 1995 SNAP-25 is expressed in islets of Langerhans and is involved in insulin release. J Cell Biol 128:1019–1028[Abstract]
  10. Jacobsson G, Bean AJ, Scheller RH, Juntti-Berggeren L, Deeney JT, Berggren P-O, Meister B 1994 Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells. Proc Natl Acad Sci USA 91:12487–12491[Abstract/Free Full Text]
  11. Oyler GA, Higgins GA, Hart RA, Battenberg I, Billinsley M, Bloom FE, Wilson MC 1989 The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations. J Cell Biol 109:3039–3052[Abstract]
  12. Niemann H, Blasi J, Jahn R 1994 Clostridial neurotoxins: new tools for dissecting exocytosis. Trends Cell Biol 4:179–185[CrossRef]
  13. Blasi J, Chapman ER, Link E, Binz T, Yamasaki S, De Camilli P, Sufhof TC, Niemann H, Jahn R 1993 Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365:160–163[CrossRef][Medline]
  14. Binz T, Blasi J, Yamasaki S, Baumeister A, Link E, Sudhof TC, Jahn R, Niemann H 1994 Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J Biol Chem 269:1617–1620[Abstract/Free Full Text]
  15. Hayashi T, Yamasaki S, Nauenburg S, Binz T, Niemann H 1995 Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro. EMBO J 14:2317–2325[Abstract]
  16. Banerjee A, Kowalchyk JA, DasGupta BR, Martin TF 1996 SNAP-25 is required for a late post-docking step in calcium-dependent exocytosis. J Biol Chem 271:20227–20230[Abstract/Free Full Text]
  17. Gutierrez LM, Viniegra S, Rueda J, Ferrer-Montiel AV, Canaves JM, Montal M 1997 A peptide that mimics the C-terminal sequence of SNAP-25 inhibits secretory vesicle docking in chromaffin cells. J Biol Chem 31:2634–2639[CrossRef]
  18. Sheng Z-H, Rettig J, Cook T, Catterall WA 1996 Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature 379:451–454[CrossRef][Medline]
  19. Wiser O, Bennett, MK, Atlas D 1996 Functional interaction of syntaxin and SNAP-25 with voltage-dependent-sensitive L- and N-type channels. EMBO J 15:4100–4110[Abstract]
  20. Lawrence GW, Weller U, Dolly JO 1994 Botulinum A and the light chain of tetanus toxins inhibit distinct stages of MgATP-dependent catecholamine exocytosis from permeabilized chromaffin cells. Eur J Biochem 222:325–333[Abstract]
  21. Kiraly-Borri CE, Morgan A, Burgoyne RD, Weller U, Wollheim CB, Lang J 1996 Soluble SNAP and NEM-insensitive factors are required for calcium stimulated exocytosis of insulin. Biochem J 314:199–203[Medline]
  22. Rajan AS, Aguilar-Bryan L, Nelson DA, Yaney GC, Ysu WH, Kunze DL, Boyd E 1990 Ion channels and insulin secretion. Diabetes Care 13:340–363[Abstract]
  23. Boyd RS, Duggan JJ, Shone CJ, Foster KA 1995 The effect of botulinum neurotoxins on the release of insulin from the insulinoma cell lines HIT-T15 and RINm5F. J Biol Chem 270:18216–1821824[Abstract/Free Full Text]
  24. Meglasson MD, Manning CD, Najafi H, Matschinsky FM 1987 Fuel-stimulated insulin secretion by clonal hamster ß cell line HIT T15. Diabetes 36:477–484[Abstract]
  25. Regazzi R, Li G, Deshusses J, Wollheim CB 1990 Stimulus-response coupling in insulin-secreting HIT cells. J Biol Chem 265:15003–15009[Abstract/Free Full Text]
  26. Nagamatsu S, Fujiwara T, Nakamichi Y, Watanabe T, Katahira H, Sawa H, Akagawa K 1996 Expression and functional role of syntaxin 1/HPC-1 in pancreatic ß cells. J Biol Chem 271:1160–1165[Abstract/Free Full Text]
  27. Bokvist K, Eliasson L, Ammala C, Renstrom E, Rorsman P 1995 Co-localization of L-type calcium channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic ß cells. EMBO J 14:50–57[Abstract]
  28. Gaisano HY, Ghai M, Malkus PN, Sheu L, Bouquillon A, Bennett MK, Trimble WS 1996 Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells. Mol Biol Cell 7:2019–2027[Abstract]
  29. Gaisano HY, Wong D, Sheu L, Foskett JK 1994 Calcium release by the cholecystokinin analog, CCK-OPE is IP3-dependent in single rat pancreatic acinar cells. Am J Physiol 265:C220–C228