Munc-18 Associates with Syntaxin and Serves as a Negative Regulator of Exocytosis in the Pancreatic β-Cell*

The Munc-18 protein (mammalian homologue of theunc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic β-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca2+ channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting β-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca2+ channels.

The Munc-18 protein (mammalian homologue of the unc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic ␤-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca 2؉ channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting ␤-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca 2؉ channels.
Exocytosis is a highly regulated process involving the docking and fusion of secretory vesicles with the plasma membrane (1)(2)(3). According to the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) 1 hypothesis of vesicle fusion, vesicles dock to a target membrane through the interaction of complementary sets of vesicular (v-SNARE) and target (t-SNARE) membrane proteins. In synaptic vesicle exocytosis, the vesicular protein synaptobrevin (also called vesicle-associ-ated membrane protein (VAMP)) is the v-SNARE, and the plasma membrane-associated proteins SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin 1 function as t-SNAREs (4 -11). Formation of the SNARE complex (or core complex) is followed by recruitment of the cytosolic proteins NSF (soluble N-ethylmaleimide-sensitive factor) and ␣-, ␤-, and ␥-SNAP (soluble NSF attachment protein), which are required for membrane fusion (12). Evidence from yeast genetics suggest that other proteins also participate in the event of exocytosis and may therefore play a role in the regulation of assembly of the SNARE complex (2). Such proteins are for instance members of the yeast Sec1 family (13,14). Sec1 homologues have been identified in the nervous system of Caenorhabditis elegans (UNC-18) (15,16), Drosophila melanogaster (ROP) (17,18), and mammals (19 -22). In mammals, the protein has been termed mammalian homologue of the unc-18 gene (Munc-18), rbSec1 (rat brain Sec1), or n-Sec1 (neural-specific Sec1) (14, 19 -22). Sec1 interacts directly with the t-SNARE syntaxin, as first suggested by genetic studies in yeast (23) and later also confirmed with the mammalian homologues (19 -22, 24). Sec1 may therefore control the presynaptic SNARE complex through an interaction with syntaxin 1 (25). The binding of Munc-18 to syntaxin inhibits the interaction of syntaxin with VAMP and SNAP-25, as well as SNAP-23 (a functional, ubiquitously expressed homologue of SNAP-25), and thereby negatively regulates the formation of the synaptic SNARE fusion complex (20, 21, 24, 26 -31). The three-dimensional structure of the neuronal Sec1-syntaxin 1A complex was recently presented (25). Mutations in Munc-18 homologues cause phenotypes associated with a complete block in neurotransmitter release and/or secretion (15,18,32,33).
Apart from the presence of Munc-18 in neuronal cells, Munc-18 mRNA has also been detected in pancreatic endocrine islet and in insulin-producing cells (38,39). In the present study, we have investigated the cellular localization of Munc-18 at the protein level in pancreatic endocrine cells using subcellular fractionation combined with Western blotting as well as immunohistochemistry combined with confocal laser microscopy. The possible association of Munc-18 with syntaxin in insulin-secreting HIT-T15 cells was investigated by means of immunoprecipitation. The effect of Munc-18 peptide and Munc-18 antiserum on insulin release was studied in streptolysin O-permeabilized insulin-secreting HIT-T15 cells. Wholecell patch clamp recordings were used to study the effect of Munc-18 peptide and Munc-18 antiserum on voltage-gated Ca 2ϩ currents.

EXPERIMENTAL PROCEDURES
Homogenate and Equilibrium Sucrose Density Gradient Preparation-HIT-T15 cells were grown to 70% confluency in 80-cm 2 Nunc TM flasks (Nunc Inc., Naperville, IL) and homogenized in the presence of protease inhibitors. The HIT-T15 cell homogenate was fractionated by equilibrium density gradient centrifugation essentially as described (40). Briefly, the cells were detached from the flasks using a 2-min incubation in PBS with 10 mM EDTA at 37°C, pelleted by centrifugation, and resuspended in homogenization buffer (4 mM HEPES, pH 7.4, 1 mM MgCl 2 , 250 mM sucrose, 0.005% DNase, and the following protease inhibitors: 4 g/ml of pepstatin A, leupeptin, antipain, and aprotinin, 0.4 mM phenylmethylsulfonyl fluoride, and 10 mM benzamidine. The cells were homogenized using 40 strokes of a Teflon/glass homogenizer. Homogenate was produced by centrifugation at 5,000 ϫ g for 10 min at 4°C. Protein estimation of HIT-T15 cell homogenate was performed by protein microassay (Bio-Rad). The homogenate of HIT-T15 cells was supplemented with EDTA to a final concentration of 1.5 mM, loaded onto a linear sucrose density gradient (0.45-2.0 M), and centrifuged at 4°C for 18 h at 100,000 ϫ g, and then seven fractions (700 l) were collected. The molarity of each fraction was determined.
SDS-PAGE and Western Blotting-18 g of protein of each HIT-T15 cell fraction and 9.4 g of brain homogenate (as a positive control) were denatured for 5 min at 100°C in SDS-PAGE sample buffer (41). An analysis was performed on an 8% (HIT-T15 cell fractions) or 7.5% (rat brain homogenate) SDS-PAGE (42). The proteins were transferred to 0.2 M nitrocellulose (Schleicher & Schuell) in 10% methanol, 20 mM Tris, 150 mM glycine, and 0.05% SDS for 12-14 h at 0.35 mA (43). Nonspecific binding of the primary antibody was blocked by incubation in 5% dried milk in buffer A (50 mM Tris, 150 mM NaCl, pH 7.6, containing 0.1% Tween 20). The blot was subsequently probed overnight at 4°C with a mouse monoclonal anti-Munc-18-1 antibody (diluted 1:700; catalog no. M32320, BD Transduction Laboratories, Lexington, KY). The blots were washed with buffer B (50 mM Tris, 150 mM NaCl, pH 7.6, containing 0.5% Tween 20) and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated donkey-anti-mouse IgG (diluted 1:5000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in buffer A and finally washed with buffer B. Detection was performed using an ECL Plus detection system (Amersham Pharmacia Biotech) and the immunoreactive bands visualized with Hyperfilm (Amersham Pharmacia Biotech).
Immunoprecipitation-Protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) was rinsed 3 times with lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100) containing protease inhibitor mixture (1:50, Roche, Basel, Switzerland). Homogenates of HIT-T15 cells and rat brain were precleared at 4°C for 2 h with protein A-Sepharose to eliminate nonspecific IgG and protein that bind nonspecifically to the beads. The beads were spun down for 2 min at 7000 ϫ g. The supernatants were incubated at 4°C overnight on a rotatory shaker with rabbit antiserum to Munc-18-1 (catalog no. 116-002, Synaptic Systems GmbH, Göttingen, Germany), or normal rabbit serum as control, followed by incubation with protein A-Sepharose at 4°C for 2 h. The beads were collected by centrifugation and washed 3 times at 4°C with lysis buffer without Triton X-100. After the final wash, the beads were resuspended in 8 l of 5ϫ SDS-PAGE sample buffer, heated for 20 min at 60°C in a water bath, and centrifuged. The supernatants were resolved by electrophoresis on 10% polyacrylamide SDS-PAGE gel and blotted using a monoclonal antibody against syntaxin 1 (diluted 1:1,000; product S-0664, clone HPC-1, Sigma).
Monolayers of the clonal insulin-secreting HIT-T15 cells were cultured in Nunc TM dishes (Nunc) using RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, streptomycin (100 g/ml), penicillin (100 units/ml), and 11 mM glucose and kept at 37°C in a humidified incubator with 95% air, 5% CO 2 (45,46). Cells were used between passages 60 and 70. The cells were fixed by immersion in fixative as described above for 15 min, rinsed with PBS (pH 7.4) for 20 min and buffer C containing 0.3% Triton X-100 for 30 min, and incubated with 5% horse serum for 1 h. The cells were incubated thereafter with rabbit antiserum to Munc-18, processed as described above, and visualized with Cy3-conjugated donkey anti-rabbit secondary antibody. Specimens were examined in a Bio-Rad Radiance Plus confocal laser scanning system. The excitation wavelength was 488 nm for fluorescein isothiocyanate-, 543 nm for lissamine-rhodamine-and Cy3-, and 638 nm for Cy5-induced fluorescence. The images were produced using a Fuji Pictrography 3000 printer.
Effect of Munc-18 Peptide and Munc-18 Antiserum on Insulin Release-HIT-T15 cells were grown in 96-well plates and incubated in a permeabilization buffer (potassium glutamate 140 mM, NaCl 5 mM, MgCl 2 1 mM, EGTA 10 mM, and HEPES 25 mM with 0.025% albumin, pH 7.0) with 1 mM dithiothreitol and 12.5 mg/ml streptolysin O (Difco) at 37°C for 8 min. The cells were incubated thereafter in the modified permeabilization buffer additionally containing 2 mM MgATP, 2 mM creatine phosphate, 10 units/ml creatine phosphokinase, and pCa 7.5 and 5 at 37°C for 20 min. In the experiments using Munc-18-1 antiserum, permeabilized cells were incubated with the modified permeabilization buffer containing Munc-18-1 antiserum (2.5 g/ml; BD Transduction Laboratories), first at 4°C for 30 min, after which the temperature was raised to 37°C. The insulin content in the buffer was measured by radioimmunoassay. The level of insulin secretion at 10 M free Ca 2ϩ was taken as 100%.
A synthetic peptide corresponding to amino acids 575-594 of rat Munc-18 (19) was synthesized (StressGen Biotechnologies, Victoria, Canada). IgG was obtained from Calbiochem. Student's t test was used for statistical analysis. After obtaining a seal, the holding potential was set at Ϫ70 mV during the course of an experiment, and depolarizing voltage pulses (70 mV, 100 ms, 0.05 Hz) were applied. The resulting currents were recorded with an Axopatch 200 amplifier (Axon Instruments, Foster City, CA), and filtered at 1 kHz. All recordings were made at room temperature (about 22°C). The integrated Ca 2ϩ currents were normalized by the capacitance of cells. Acquisition and analysis of data were done using the software program pCLAMP (Axon Instruments).

Subcellular Fractionation and Western
Blotting-Incubation of rat brain homogenate with a mouse monoclonal antibody raised against Munc-18-1 revealed a strong band of approximately 67 kDa (Fig. 1). In subcellular fractions of HIT-T15 cells, there were bands corresponding to the predicted size of 67 kDa in all of the seven subcellular fractions, including both cytosol (Fig. 1, fractions 1 and 2) and membrane (fractions 4 -6) fractions. Fraction 3 represents an intermediate of membrane and cytosol fractions, whereas fraction 7 represents a nuclear fraction. At a short exposure time, it was evident that there were two bands at around the predicted size of 67 kDa in rat brain homogenate as well as in fractions 2-6 but not in fractions 1 and 7.
Immunoprecipitation-To evaluate the possible association of Munc-18-1 with syntaxin 1, immunoprecipitation was performed on homogenates of HIT-T15 cells and rat brain using a polyclonal antibody against Munc-18-1. A 35-kDa band corre-sponding to syntaxin 1 was demonstrated in extracts of HIT-T15 cells and rat brain (Fig. 2), which had been immunoprecipitated using the Munc-18-1 antiserum.
Immunohistochemistry-There was strong Munc-18 immunoreactivity in virtually all cells of the islets of Langerhans as well as in cultured HIT-T15 cells (Figs. 3A-C, and 4, A, C, E, and G). Munc-18 immunoreactivity was also demonstrated in nerve fibers and nerve terminals present around blood vessels and extending into the islets and the acini of exocrine cells (Fig.   FIG. 1. Expression of Munc-18 protein in rat brain and in subcellular fractions from insulin-producing HIT-T15 cells detected by immunoblotting using a mouse monoclonal antibody to Munc-18. Equal amounts of protein from total rat brain homogenate (9.4 g) and subcellular fractions from HIT-T15 cells (18 g each) were added to the gel. Fractions: 1-2, cytosol; 3, intermediate of fractions 1-2 and 4 -6; 4 -6, membrane; 7, nuclear. A protein corresponding to the expected size of 67 kDa was detected in rat brain and in cytosol as well as in membrane fractions of HIT-T15 cells. The 67-kDa bands of fractions 2-6 of HIT-T15 cells consist of two distinct bands. The experiment was repeated four times.

FIG. 2. Association of Munc-18 with syntaxin 1 in rat brain and
HIT-T15 cells. 12.5 g of protein from rat brain homogenate and 150 g of protein from HIT-T15 cell homogenate were subjected to immunoprecipitation using a rabbit polyclonal antiserum to Munc-18 or normal rabbit serum. A 35-kDa band corresponding to syntaxin 1 is demonstrated in extracts of rat brain and HIT-T15 cells that have been immunoprecipitated with Munc-18 antiserum (lane 1) and in homogenates of rat brain and HIT-T15 cells (lane 3). No syntaxin 1 immunoreactivity is detected in rat brain and HIT-T15 cell homogenates that have been incubated with normal rabbit serum (lane 2). The experiment was repeated four times. A). Within individual cells, the immunofluorescence is punctate (B and C). Bars ϭ 10 m.

FIG. 3. Images of sections of rat endocrine pancreas (A and B) and cultured HIT-T15 cells (C) obtained via confocal laser microscopy after incubation with rabbit antiserum to Munc-18. Strong Munc-18 immunoreactivity is present in virtually all cells of the islets of Langerhans (A and B) as well as in individual HIT-T15 cells (C). Note the Munc-18 immunoreactive nerve fibers and nerve endings between islet cells (arrows in
3A). Weak Munc-18 immunoreactivity was also demonstrated in pancreatic exocrine cells (Figs. 3A and 4, A, C, E, and G). Confocal laser microscopy revealed that Munc-18 immunoreactivity was punctate within the cytoplasm of endocrine cells (Figs. 3, A-C, and 4, A, C, E, and G).
Direct double-labeling immunofluorescence histochemistry of rat pancreatic islets demonstrated Munc-18 immunoreactiv-ity in insulin (compare Fig. 4, A with B)-, glucagon (compare Fig. 4, C with D)-, pancreatic polypeptide (compare Fig. 4, E with F)-, and somatostatin (compare Fig. 4, G with H)-containing cells. Incubation with non-immune serum did not reveal any fluorescence in the rat pancreas (data not shown).
Effects there was no significant difference in insulin release from streptolysin O-permeabilized HIT-T15 cells after the addition of Munc-18 peptide-(575-594) (25 M) (19) in comparison with control (Fig. 5A). However, in the presence of 10 M Ca 2ϩ , there was a significant increase in insulin release after administration of Munc-18 peptide (25 M) compared with control (Fig.  5A). The level of insulin secretion at 10 M free Ca 2ϩ was taken as 100%.
Administration of rabbit Munc-18 antiserum (2.5 mg/ml) or normal rabbit IgG (2.5 mg/ml) did not alter insulin release in the presence of 30 nM Ca 2ϩ in permeabilized HIT-T15 cells as compared with control (Fig. 5B). However, there was a significant increase in insulin release after the addition of Munc-18 antiserum (2.5 g/ml) as compared with control in the presence of 10 M Ca 2ϩ , which was not detected after addition of normal rabbit IgG (2.5 g/ml) alone (Fig. 5B).

Effects of Munc-18 Peptide and Munc-18 Antiserum on Voltage-gated Ca 2ϩ Channel Activity in Single HIT-T15 Cells-To
test for the possible modulation of voltage-gated Ca 2ϩ currents by Munc-18, 25 M synthetic Munc-18 peptide-(575-594) (19) and a polyclonal antibody (1:100) against Munc-18-1 (Synaptic Systems) were perfused into cells during recordings of wholecell Ca 2ϩ currents. Typical Ca 2ϩ current traces evoked by repetitive depolarizing voltage pulses from a holding potential of Ϫ70 to 0 mV (100 ms, 0.05 Hz) were not significantly different between control cells and cells perfused with Munc-18 peptide, as shown by the 15 superimposed current traces in Fig. 6A. The compiled data, as illustrated in Fig. 6B, show that integrated Ca 2ϩ currents ran up similarly in cells treated with Munc-18 peptide as compared with control cells. There was no significant difference in integrated Ca 2ϩ currents between the two groups. However, the modulatory effects of Munc-18 on voltage-gated Ca 2ϩ channels could not be excluded, because synthetic Munc-18 peptide may bind to syntaxin 1. Therefore, the complexes of syntaxin 1 and synthetic Munc-18 peptide or native Munc-18 may have similar effects on voltage-gated Ca 2ϩ channels. To block the binding of Munc-18 to syntaxin 1, a polyclonal Munc-18 antiserum was perfused into HIT-T15 cells. Perfusion of HIT-T15 cells with Munc-18 antiserum or normal rabbit IgG did not reveal any significant differences in the Ca 2ϩ current run-up and the amount of voltage-dependent Ca 2ϩ influx (Fig. 6, C and D). DISCUSSION Our results show that the Munc-18 protein is present in pancreatic ␤-cells, that Munc-18 is associated with the t-SNARE syntaxin in insulin-producing cells, and that Munc-18 has a regulatory role in insulin exocytosis via a mechanism not involving voltage-gated Ca 2ϩ channels. This study adds further data to the increasing evidence suggesting that exocytotic proteins, previously shown to be of vital importance for neuronal exocytosis, are also essential components of the insulin exocytotic machinery.
The Munc-18 protein has previously been shown to be present in a soluble and a membrane-associated form (28), the latter concentrated in the plasma membrane and in large vesicles associated with the Golgi apparatus (47). In agreement with these findings, our results from the subcellular fractionation of insulin-producing HIT-T15 cells revealed a 67-kDa Munc-18 protein in both cytosol and membrane fractions. Two distinct bands around the predicted size of 67 kDa were detected mainly in the membrane fractions, which may reflect the presence of different Munc-18 variants in insulin-secreting cells. The antisera used in this study have been generated to Munc-18-1, implicating that the Munc-18 immunoreactivity detected by both immunoblotting and immunohistochemistry represents Munc-18-1 protein.
A negative regulatory role of n-Sec1/Munc-18 in exocytosis has been suggested based on biochemical data (24). In vitro, n-Sec1 inhibits SNAP-25 and VAMP binding to syntaxin, thereby inhibiting the formation of the core complex (24). In agreement with a negative regulatory role for n-Sec1/Munc-18 in neurotransmitter release are results showing that microinjection of Sec1 into the presynaptic terminal of the giant squid synapse inhibits evoked transmitter release (30). Overexpression of ROP, the Drosophila homologue of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins, reduces neurotransmitter release in a dose-dependent fashion in vivo via an interaction with syntaxin (27). Mutations of Munc-18 homologues in Drosophila (ROP), C. elegans (unc-18), and yeast (Sec1), as well as in Munc-18-1, all result in phenotypes with blocked neurotransmitter release (15,18,48,49). Recent observations using point mutations in ROP suggest that ROP is a rate-limiting regulator of exocytosis that performs both stimulatory and inhibitory functions in neurotransmission (50). The reduction in neurotransmitter release seen after both overexpression of Munc-18 and mutations in Munc-18 homologues indicates that Munc-18 proteins not only sequester syntaxins from other proteins but also assist the syntaxins in adopting a functional conformation or facilitate interactions between syntaxins and other proteins by a chaperone-like action. Syntaxin has been shown to be required for a post-docking role (51), and because ROP interacts with syntaxin, it has been concluded that the function of ROP is mediated after the docking step (50). Taken together, the evidence supports the opinion that Munc-18 and other homologues of Sec1 play negative roles in neurotransmitter release (14,35).
The binding of Munc-18 to syntaxin 1 prevents formation of the SNARE complex (24,52), and syntaxin 1 bound to Munc-18 adopts a closed conformation in which the N-terminal of the protein interacts with its own C-terminal. The removal of Munc-18 from syntaxin 1 results in opening of this closed conformation and promotes formation of the SNARE complex. In this study, manipulation with Munc-18 in permeabilized cells modulated exocytosis of insulin. First, introduction of Munc-18 antibody into permeabilized cells produced an increase in Ca 2ϩ -induced exocytosis. Second, a 20-amino acid C-terminal peptide of the protein also stimulated Ca 2ϩ -dependent exocytosis. Introduction of antibody most likely forced the dissociation of the Munc-18⅐syntaxin 1A complex by extracting Munc-18 with the antibody. Recent data on the Munc-18⅐syntaxin 1A complex indicate that the C terminus of Munc-18 forms domain 2 of the protein (25). This domain is not involved in the interaction with syntaxin 1 protein. Hence, it is difficult to envisage that introduction of the C-terminal peptide into the HIT-T15 cells would directly interfere with the Munc-18⅐syn-taxin 1 interaction. Stimulation of exocytosis with the peptide probably suggests that domain 2 of Munc-18 is important for the interaction of Munc-18 with other proteins. Sec1 family members also interact with Rab proteins, which are small GTPases that also are essential for vesicle trafficking and membrane fusion (53). Disturbance of Rab proteins could be the reason for the stimulation of insulin exocytosis by the Munc-18 C-terminal peptide.
Syntaxin 1 contains a helical, autonomously folded N-terminal domain, a C-terminal SNARE motif, and a transmembrane region. The SNARE motif binds to SNAP-25 and VAMP to assemble the core complex, whereas most of the cytoplasmic region participates in a complex with Munc-18-1 (54). The interaction of Munc-18 with syntaxin 1 has been well characterized in vitro (21,24) and in vivo (30,55), and more recently, the three-dimensional structure of the Munc-18⅐syntaxin 1A complex has been described (25). Our results show that Munc-18 is present in insulin-producing pancreatic cells and that syntaxin and Munc-18 were coimmunoprecipitated in insulin-producing HIT-T15 cells. These results combined show that there is an association of Munc-18 with syntaxin in ␤-cells.
Apart from being a regulator of neurotransmitter release, syntaxin 1 plays a central role in insulin exocytosis (56 -58). Previous studies have shown that the H3 region of the syntaxin 1 C-terminal end interacts with the ␣ 1B subunit of the N-type Ca 2ϩ channel in a Ca 2ϩ -dependent manner (59,60). In pancreatic ␤-cells, syntaxin 1 interacts with the L C and L D subtypes of voltage-gated Ca 2ϩ channels (61,62). It should therefore be considered that the binding of Munc-18 to syntaxin may indirectly interfere with Ca 2ϩ channels in ␤-cells and may thereby affect insulin secretion. However, our results did not reveal any effect of Munc-18 peptide or antiserum on voltage-gated Ca 2ϩ channel activity. These negative findings indicate that Munc-18 functions as a negative regulator of insulin secretion through intervention of interaction between v-SNARES and t-SNARES, without interfering with syntaxin or voltage-gated Ca 2ϩ channels. The exact mechanism by which Munc-18 affects insulin exocytosis remains to be established.