D.-H. Kweon, School of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, Korea Fax: +82 31 290 7870 Tel: +82 31 290 7869 E-mail: firstname.lastname@example.org
Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins have central roles in neurotransmission. At the synapse, membrane fusion, which is required for neurotransmitter release, is mediated by SNAREs. Vesicle-associated membrane protein 2 (v)-SNARE synaptobrevin (VAMP2) associates with target membrane (t)-SNAREs syntaxin 1a and synaptosome-associated protein of 25 kDa (SNAP-25) [1–3] to form the highly stable ternary SNARE complex [4–6]. Cumulative evidence has shown that the SNARE complex forms the core of the machine that generates the energy required for membrane fusion, while other accessory proteins are involved in docking, tethering, Ca2+-sensing and recycling. A fusion pore is created as a result of membrane fusion and neurotransmitters are released through this pore [7–12].
The SNARE complex is a parallel four-helical bundle [4,5]. The four core helices are provided by the three SNARE proteins; one each from syntaxin and VAMP2, and two from SNAP-25 [4,5]. Syntaxin and VAMP2 are transmembrane proteins with single transmembrane helices and are anchored to the presynaptic membrane and vesicular membrane, respectively. SNAP-25 is peripherally attached to the presynaptic membrane. Thus, formation of a parallel coiled-coil would result in close apposition of two membranes, and facilitate membrane fusion. The core complex has been shown to be linked tightly to the membranes so that the force generated by SNARE complex formation can be faithfully transferred to the membranes [13–15].
Impaired SNARE function is known to block neuronal exocytosis. For example, synaptic SNARE proteins are the specific substrates of eight clostridial neurotoxins (one tetanus neurotoxin and seven botulinum neurotoxins; BoNT/A–G) [16,17]. The neurotoxins bind specifically to the nerve terminals and deliver the N-terminal catalytic domain of the zinc-endopeptidase into the cytosol, where the catalytic domain specifically cleaves a SNARE protein at a single site within its cytosolic portion. Such specific cleavage leads to an inhibition of neuroexocytosis and results in the paralytic syndromes of botulism and tetanus. Low doses of BoNT/A are now widely used to alleviate the symptoms of various disorders, including paralytic strabismus, blephoraspasm, cervical dystonia and severe hyperhydrosis [18,19]. The neurotoxin is also widely used for cosmetic purposes. Clostridial toxins have proven very versatile with therapeutic and cosmetic uses.
Inhibition of neurotransmission might be achieved not only by the specific cleavage of SNARE proteins, but also by blocking SNARE complex formation, and several peptide inhibitors have been developed for this purpose [20–27]. The peptides are mostly modeled on the sequences of the SNARE motifs in SNAP-25. These peptides are thought to competitively inhibit SNARE complex formation by interfering with interactions between parental SNARE proteins. However, no systematic study has evaluated the efficacy of SNARE-patterned sequences from all four SNARE motifs. As such, there is no design principle to guide the development of potent peptide SNARE inhibitors. Because individual sequences modeled on the SNARE motifs are expected to inhibit SNARE complex formation to differing degrees, careful assessment of the effect of SNARE-patterned peptides on SNARE complex formation might help us better understand SNARE-driven neuronal exocytosis. Such efforts will also help us identify the potent peptide sequences that interfere with neurotransmission. In this study, we designed and synthesized small peptides derived from SNARE motifs. We assessed their inhibitory activities on membrane fusion, neuroexocytosis of digitonin-permeabilized PC12 cells and SNARE complex formation in PC12 cells. We found that the peptide sequences derived from the N-terminal regions of SNARE motifs show high inhibitory activity.
Design of peptides modeled on the SNARE motifs
Peptide sequences modeled on SNARE motifs are most likely to compete with parental SNARE proteins for SNARE complex formation, inhibiting SNARE assembly. In an initial attempt to search for the potent peptide sequences that inhibit SNARE complex formation and neurotransmitter release, we synthesized thirteen 17-mer peptides representing the various regions in the SNARE motifs of individual SNAREs. Three peptide sequences were derived from each SNARE motif, giving 12 from four SNARE motifs. Three sequences from each SNARE motif represent the N-terminal, middle and C-terminal regions (Fig. 1A). Sequences representing the middle regions contained the amino acid Q or R that is present in the zero-layer of the SNARE complex (bold italics in Table 1) . Individual peptide sequences are shown in Table 1. One additional 17-mer peptide (designated as LIB), which was previously selected as a SNARE inhibitor from an α-helix-constrained combinatorial peptide library , was prepared as a positive control.
Table 1. Amino acid sequences of synthetic peptides. Glutamine and arginine residues in the zero layer are shown in bold italics.
SNN (7–23) MRNELEEMQRRADQLAD
SNM (45–61) RTLVMLDEQGEQLERIE
SNC (65–71) DQINKDMKEAEKNLTDL
SCN (140–156) DARENEMDENLEQVSGI
SCM (166–182) DMGNEIDTQNRQIDRIM
SCC (190–206) TRIDEANQRATKMLGSG
SynN (192–208) LSEIETRHSEIIKLENS
SynM (218–224) DMAMLVESQGEMIDRIE
SynC (245–261) ERAVSDTKKAVKYQSKA
VpN (30–46) RRLQQTQAQVDEVVDIM
VpM (48–64) VNVDKVLERDQKLSELD
VpC (76–92) QFETSAAKLKRKYWWKN
Inhibition of SNARE-driven membrane fusion by synthetic peptides
In order to measure the efficacy of 13 synthetic peptides, a fluorescence lipid-mixing assay was performed using SNARE-reconstituted liposomes (see Experimental procedures) [3,28]. The lipid-mixing assay is a well-established method that has frequently been used to show various features of SNARE-driven membrane fusion [7,12,28–33].
For the lipid-mixing assay, full-length t-SNARE complex was reconstituted into large unilamellar vesicles (100 nm diameter) composed of 1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) in a molar ratio of 65 : 35. Full-length v-SNARE was reconstituted into a separate population of the same POPC/DOPS vesicles containing 1.5 mol% each of fluorescence lipids. When the t-SNARE and v-SNARE vesicles were mixed without adding the synthetic peptides there was an increase in the fluorescence signal (Fig. 2A), indicating that lipid mixing had occurred.
Several synthetic peptides showed rather poor solubility in water, whereas others had good solubility in buffer. Therefore, dimethylsulfoxide was used to dissolve peptides with poor water solubility and peptide/dimethylsulfoxide solutions were injected directly into the fusion reaction. The added dimethylsulfoxide influenced lipid mixing somewhat (Fig. 2A); therefore, the inhibition efficacies of the peptides were tested in two different batches depending on the solvent conditions (Fig. 2).
Some synthetic peptides had profound effects on membrane fusion when tested at a peptide concentration of 200 μm (Fig. 2A,B). Peptides representing the N-terminal region of the SNARE motifs SCN (from SNAP-25C), SynN (from Syntaxin 1a) and VpN (from VAMP2) reduced membrane fusion significantly, whereas the other peptides were much less effective. The most effective peptides (VpN and SynN) decreased membrane fusion by as much as 60–70% of the control (Fig. 2B).
The amphipathic character of the SNARE-patterned peptides may make them fusogenic [34–36]. In order to rule out this possibility, the fusion activities of the peptides were tested in the absence of SNAREs (Fig. 2B, gray bar). None of the 17-mer peptides had significant membrane fusion activity without SNAREs. The cytoplasmic domain of VAMP2 (VpS, amino acids 1–96) has been used frequently as an inhibitor for SNARE-driven membrane fusion . VpS suppressed membrane fusion very effectively at lower concentrations (20 μm) (Fig. 2A,B) and served as a good positive control to compare the inhibitory effect of the synthetic peptides.
In order to verify whether the reduced membrane fusion was caused by inhibition of SNARE complex formation by the peptides, western blot analysis was performed for six peptides after membrane fusion. The SDS-resistant SNARE complex was detected using anti-(SNAP-25) IgG. SCN, SynN and VpN reduced SNARE complex formation, whereas SNN, SCM and LIB were not effective (Fig. 2C), supporting the idea that the inhibitory activities were due to inhibition of SNARE complex formation by the peptides.
Interestingly, SCM increased membrane fusion by ∼ 70% (Fig. 2A,B), although it did not enhance SNARE complex formation (Fig. 2C). It has previously been shown that a synthetic peptide from the C-terminal region of VAMP2 promoted SNARE-mediated fusion . Pobbati et al.  showed that this stimulatory effect might be due to its role in refolding the t-SNARE complexes into an active conformation. We wonder whether SCM has a similar effect in SNARE-mediated membrane fusion, warranting further investigation.
Inhibition of neurotransmitter release from PC12 cells by the synthetic peptides
In order to measure the effects of synthetic peptides on neuronal exocytosis, we prepared permeabilized PC12 cells loaded with [3H]-noradrenaline. Depolarization of these cells by high K+ concentrations (50 mm KCl) results in the release of neurotransmitters such as noradrenaline, acetylcholine and arachidonic acid [38,39]. Inhibition of this release by synthetic peptides could be assessed by measuring [3H]-noradrenaline release after stimulation with high concentrations of K+, in the presence or absence of synthetic peptides. In the permeabilized PC12 cells, stimulation with high concentrations of K+ significantly increased [3H]-noradrenaline release, compared with the basal levels. We tested the effects of the synthetic peptides on the high K+ concentration-stimulated release of [3H]-noradrenaline. Addition of SCN, SynN and VpN efficiently inhibited neuroexocytosis even at a final concentration of 10 μm, whereas other peptides showed no significant effect (Fig. 3A), consistent with results from the lipid-mixing assay. Inhibition of [3H]-noradrenaline release by the N-terminal-mimicking peptides SCN, SynN and VpN was as much as 20–30%, when compared with controls (Fig. 3A). Verapamil, an L-type calcium-channel blocker, inhibited neurotransmitter release by ∼ 70% at the same concentration (10 μm) [40,41]. Thus, N-terminal-mimicking peptides serve as good SNARE-targeting neuroexocytosis inhibitors. We were not able to test higher peptide concentrations because some peptides were dissolved in dimethylsulfoxide which killed PC12 cells at higher concentrations.
In order to verify whether the peptides inhibited SNARE assembly in PC12 cells, membrane proteins were extracted from the cells and analyzed by western blotting using anti-(SNAP-25) IgG (Fig. 3B,C). Consistent with other results, SCN, SynN and VpN inhibited SNARE complex formation significantly (Fig. 3B,C). VpN inhibited SNARE complex formation with a similar efficiency to verapamil. N-Terminal-mimicking peptides inhibited SNARE complex formation by directly interacting with native SNARE proteins, whereas verapamil did the same by blocking calcium entry. By contrast, other peptides did not have noticeable inhibitory effects on SNARE complex formation. Therefore, the results show that the reduction in neuroexocytosis by SCN, VpN and SynN was a direct consequence of the inhibition of SNARE complex formation.
Smaller peptides might be beneficial in terms of synthesis costs, as well as membrane penetration. Therefore, we synthesized nine additional 6-residue peptides derived from v-SNARE VAMP2 (Fig. 1A and Table 1). We also made the well-known Argireline [26,42]. We did not observe any measurable inhibitory effects with the 6-mer peptides when tested using the lipid-mixing assay (data not shown) and noradrenaline release assay (Fig. 3).
Time-dependent effects of inhibitory peptides on membrane fusion and neurotransmitter release
N-Terminal zippering may be the rate-limiting step in SNARE complex formation. The N-terminal-mimicking peptides may have inhibited this N-terminal zippering, resulting in the most significant inhibition of membrane fusion and neurotransmitter release. Conversely, N-terminal-mimicking peptides might not inhibit SNARE complex formation and membrane fusion when partial N-terminal zippering has occurred. In order to test this, five selected synthetic peptides were added to the membrane fusion reaction mixture before or after preincubation of t- and v-SNARE vesicles at 4 °C overnight . SNN and SCC were selected as negative controls because they did not inhibit membrane fusion and neurotransmitter release (Figs 2 and 3).
Preincubation of t- and v-SNARE vesicle mixture at 4 °C overnight accelerated the membrane fusion reaction by ∼ 40% when the reaction was induced by increasing the temperature to 37 °C, consistent with previous results (Fig. 4A; upper left) . When SCN, SynN and VpN were added before preincubation, membrane fusion was consistently inhibited. However, the inhibitory effect of those peptides was much lower when they were added after preincubation. SNN and SCC did not inhibit membrane fusion, regardless of preincubation. Thus, N-terminal-mimicking peptides seem to inhibit membrane fusion by hindering N-terminal zippering. When membrane fusion was complete, the amount of SNARE complex was measured by western blotting (Fig. 4B). SNN and SCC did not change the amount of SNARE complex formed, regardless of preincubation. However, there was a dramatic change in the amount of SNARE complex depending on the addition time of SCN, SynN and VpN. When SCN, SynN and VpN were added after preincubation they did not reduce SNARE complex formation much. By contrast, these peptides inhibited SNARE complex formation very efficiently when added before preincubation. Therefore, N-terminal-mimicking peptides inhibited SNARE complex formation by hindering N-terminal zippering, which is consistent with the membrane fusion result.
These experiments show that N-terminal-mimicking peptides do not inhibit membrane fusion if the N-terminals of t- and v-SNARE proteins are already zipped together, suggesting that the target of the peptides is newly forming SNARE complex. In order to confirm this in PC12 cells, already-loaded neurotransmitters were depleted by pretreating with high concentrations of K+ before the addition of inhibitory peptides. After pretreatment with high concentrations of K+, inhibitory peptides were added and cells were cultured for 24 h to regenerate synaptic vesicles. By doing so, we are able to measure noradrenaline release from the newly docking vesicles only. In Fig. 4C, it is clearly shown that the noradrenaline release is dramatically reduced by N-terminal-mimicking peptides after pretreatment with high K+ concentrations. SCN, SynN and VpN reduced noradrenaline release to the level of verapamil, which may represent the minimum level of stimulated neuroexocytosis. SNN and SCC did not inhibit noradrenaline release regardless of pretreatment with high K+ concentrations. Verapamil, a calcium-channel blocker, did not have an additional inhibitory effect on noradrenaline release. Also, we were able to confirm that SNARE complex formation was dramatically reduced by N-terminal peptides, whereas other peptides were almost neutral with regard to SNARE complex formation in PC12 cells (Fig. 4D). In conclusion, N-terminal peptides inhibit newly forming SNARE complex in PC12 cells and block the regeneration of neuroexocytosis in synaptic vesicles. We speculate that the neurotransmitter release and SNARE complex band shown in Fig. 3 were the sum of effects derived from partially zipped SNARE complex and newly forming complex. By eliminating the effect derived from already-zipped complex, the effect of N-terminal peptides on SNARE complex formation and neuroexocytosis could be more distinctive.
Depolarization of PC12 cells induces the influx of Ca2+, leading to the fusion of synaptic vesicles which are docked at the active zone of neurotransmitter release . When the PC12 cells were depolarized with a short-pulse high concentration of K+, the synthetic peptides derived from the N-terminal regions of the SNARE motifs SCN, SynN and VpN inhibited [3H]-noradrenaline release in detergent-permeabilized PC12 cells (Fig. 3). These results correlate strongly with the reduction in SNARE complex formation in the PC12 cells (Fig. 3B,C), suggesting that the peptides inhibit [3H]-noradrenaline release by competing with parental SNAREs for complex formation.
Recently, it has been shown that N-terminal nucleation of the SNARE complex can promote rapid membrane fusion . Conversely, one could predict that the disruption of N-terminal nucleation would affect the rate of membrane fusion significantly (route A in Fig. 5). A variety of experiments including the lipid-mixing assay, the neurotransmitter release assay in PC12 cells and western blot analysis consistently confirmed that SNARE-mediated fusion is profoundly affected by N-terminal patterned peptides. Therefore, it appears that the N-terminal regions of SNARE motifs are the potential targets for developing potent blockers of SNARE-mediated membrane fusion.
The relative inefficiency of other peptides in inhibiting fusion can be rationalized in the context of the zipper model for SNARE complex formation [31,44–47]. The zipper model predicts that SNARE assembly starts in the N-terminal region and proceeds towards the C-terminal region. One may envisage that a peptide bound to the middle or C-terminal region of t-SNARE complex (route C in Fig. 5) is peeled off easily as zippering progresses , resulting in no or only moderate effects on membrane fusion (Figs 2–4).
A current model for SNARE assembly predicts that VAMP2 in synaptic vesicles associates with the binary complex of t-SNARE Syntaxin 1a and SNAP-25 in presynaptic membranes. Thus, it is likely that VAMP2-patterned peptides bind to the t-SNARE complex and compete with VAMP2 for binding. Because Syntaxin 1a and SNAP-25 can form a complex with 2 : 1 stoichiometry, it is likely that syntaxin-patterned peptides also have their binding sites within the t-SNARE complex. When SynN is bound to the 1 : 1 t-SNARE complex, however, the structure of the t-SNARE complex might change to that of the 2 : 1 complex, which is known to be a dead-end product (route D in Fig. 5) . Such a conformational change would definitely inhibit the association with VAMP2, and thus membrane fusion.
By contrast, N- and C-terminal-mimicking peptides from SNAP-25 (SNN and SCN, respectively) had rather intricate effects on neuroexocytosis (Figs 2–4). SNN did not inhibit SNARE complex formation at all. SNN might not bind to any of the SNARE complexes involved in the SNARE assembly pathway. However, SCN was as effective as SynN and VpN. SCN may bind to the partially assembled t-SNARE complex in which the C-terminal SNARE motifs of SNAP-25 are not yet engaged , competing with the binding of the C-terminal motif of SNAP-25.
The ineffectiveness of several well-known peptides that are known to regulate neuronal exocytosis is interesting. For example, a peptide mimicking the C-terminal domain of SNAP-25 (corresponding to SCC in this study) reportedly blocked Ca2+-dependent exocytosis in chromaffin cells with an IC50 of 20 μm . However, we did not observe such an inhibitory effect of SCC on SNARE assembly and neuroexocytosis in PC12 cells (Figs 2–4). In addition, LIB, which was selected from an α-helical peptide library of 137 180 sequences, was also not effective in the lipid-mixing assay or the noradrenaline release and SNARE complex formation assays in PC12 cells (Figs 2 and 3). Previously, LIB was screened by measuring the SDS-resistant complex formation with the purified SNARE proteins. We speculate that the discrepancy between our results and those found previously on the efficacies of SCC and LIB arises from differences in the measurement scale of the assays used. In a screening process, determining the selection criteria depends on the level of the background and experimental conditions. As such, a potential inhibitory peptide screened from an experiment may not necessarily be reproduced in other experiments using different assays or differently prepared cells. Therefore, a fair comparison of the effectiveness of different SNARE-patterned peptides can only be made under identical experimental conditions. In this study, the 13 representative peptides were tested under the same experimental conditions and their relative inhibition efficacies were ranked. Our results showed that SCN, SynN and VpN are much more effective than any other SNARE-patterned peptides.
It is also noteworthy that we were able to reproduce part of the previous results with LIB. When LIB was tested for its inhibitory effect on SDS-resistant complex formation of purified cytoplasmic domains of SNARE proteins in vitro it reduced SNARE complex formation by ∼ 80% at 200 μm (data not shown). This is comparable with previous results in which LIB inhibited SNARE complex formation by ∼ 95% at 0.5 mg·mL−1 (∼ 250 μm) . However, LIB did not show any significant effects on membrane fusion and neurotransmitter release in our study (Figs 2 and 3). Interestingly, the inhibition of SNARE complex formation by SCN, SynN and VpN was less than that by LIB when purified SNAREs were used (data not shown), although these peptides were much more potent in inhibiting membrane fusion and release in PC12 cells. These results suggest that the peptides screened by SDS-resistant complex formation using purified SNARE proteins might not correlate well with the inhibition of neuroexocytosis. There is evidence that the membrane has crucial roles in SNARE complex formation and membrane fusion [14,50]. Membranes may also restrict the geometry in which SNAREs assemble. Thus, inhibition of SDS-resistant complex formation of purified soluble proteins does not necessarily mirror the inhibition of membrane-anchored SNARE complex formation.
Because neuronal exocytosis is triggered on a millisecond time scale, synaptic vesicles are believed to be present at the active zone in a ready-to-fire state. However, we do not know in which state SNAREs are arrested before formation of the fusion pore: at the free protein stage, when N-terminal tips are already zipped or when the full complex is formed but the fusion pore is not yet made. In Fig. 4, we show that SCN, SynN and VpN inhibited N-terminal zippering and that the already-zipped complex was not inhibited by those peptides (Fig. 4A,B). Together with the fact that N-terminal peptides target newly forming SNARE complex (Fig. 4C,D), we might rule out the possibility that SNARE proteins are arrested before fusion at the state of free proteins. In conclusion, the inhibitory effects of the N-terminal-mimicking peptides on neurotransmitter release are very promising in the context of their applications in pharmaceuticals and cosmetics.
POPC, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphoserine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). RPMI-1640, penicillin–streptomycin, horse serum and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY, USA). Triton X-100, 2-mercaptoethanol and all other chemicals were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). All peptides used (Fig. 1A) were synthesized by Peptron, Inc. (Dajeon, Korea), and were ≥ 95% pure as judged by MS. Synthetic peptides were dissolved in distilled water or dimethylsulfoxide depending on their solubility. Argireline  and LIB  were also synthesized as controls.
Expression and purification of recombinant protein preparation
Protein expression and purification procedures for neuronal SNARE proteins have been described previously . In brief, the pGEX-2T vector encoding a thrombin-cleavable N-terminal glutathione S-transferase tag was used for the expression of the following constructs: full-length syntaxin 1a (amino acids 1–288), full-length VAMP2 (amino acids 1−116) and SNAP-25 (amino acids 1−206). All recombinant proteins were expressed in an Escherichia coli CodonPlusRIL (DE3) (Novagen, Darmstadt, Germany). All N-terminal glutathione S-transferase-tagged fusion proteins were purified by affinity chromatography using glutathione–agarose beads. The protein was cleaved by thrombin in cleavage buffer (50 mm Tris/HCl, 150 mm NaCl, pH 8.0). We added 1%n-octyl-d-glucopyranoside for syntaxin 1a and VAMP2. Purified proteins were examined by 12.5% SDS/PAGE, and the purity was at least 90% for all proteins (Fig. 1B).
Reconstitution of SNARE proteins into membranes
Large unilamellar vesicles with a diameter of 100 nm were prepared as described previously . In brief, a mixture of POPC and DOPS (molar ratio of 65 : 35) in chloroform was dried in a vacuum and resuspended in buffer (50 mm Tris/HCl, 150 mm NaCl, pH 8.0) for a total lipid concentration of 50 mm. Protein-free large unilamellar vesicles (∼ 100 nm dia.) were prepared by extrusion through polycarbonate filters (Avanti Polar Lipids Inc., Alabaster, AL, USA). Syntaxin 1a and SNAP-25 were mixed at room temperature for ∼ 60 min to allow the formation of t-SNARE complex. The t-SNARE complex was then mixed with the prepared large unilamellar vesicles at a 50 : 1 lipid/protein molar ratio. For the v-SNARE vesicles, 10 mm fluorescent liposomes, containing POPC, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphoserine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and rhodamine-PE at a molar ratio of 62 : 35 : 1.5 : 1.5, were mixed with VAMP2 at a 50 : 1 lipid/protein ratio. The liposome/protein mixture was diluted twice, which brought the concentration of n-octyl-d-glucopyranoside below the critical micelle concentration. After dialyzing against dialysis buffer (25 mm Hepes, 100 mm KCl, 5% w/v glycerin, pH 7.4) at 4 °C overnight to remove detergent, the sample was treated with Bio-Beads SM2 (Bio-Rad, Hercules, CA, USA) to eliminate any remaining detergent. The solution was then centrifuged at 10 000 g for 30 min to remove protein and lipid aggregates. The final t-SNARE liposome solution contained ∼ 2.5 mm lipids and 1.9 mg·mL−1 of protein, and the v-SNARE liposome solution contained ∼ 1 mm lipids and 0.25 mg·mL−1 of protein. The reconstitution efficiency was determined using SDS/PAGE. The amount of protein in the liposomes was estimated by comparing the band in the gel with that of a known concentration of the same protein.
SNARE-driven membrane fusion assay
The total lipid-mixing fluorescence assay has been described elsewhere . To measure total lipid mixing, v-SNARE liposomes were mixed with t-SNARE liposomes at a ratio of 1 : 9. The final solution contained 1 mm lipids, in a total volume of 100 μL, in the presence of synthetic peptide. Fluorescence was measured at excitation and emission wavelengths of 465 and 530 nm, respectively. Changes in fluorescence were recorded with a Spectra Max M2 (Molecular Devices Inc., Palo Alto, CA, USA) fluorescence spectrophotometer. The maximum fluorescence intensity was obtained by adding Triton X-100. All lipid mixing experiments were carried out at 37 °C.
PC12 cell culture
PC12 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were plated onto poly-(d-lysine)-coated culture dishes and were kept in RPMI-1640 containing 100 μg·mL−1 streptomycin, 100 U·mL−1 penicillin, 2 mm l-glutamine, 10% heat-inactivated horse serum and 5% fetal bovine serum at 37 °C in a 5% CO2 incubator. Cell cultures were split once a week, and the medium was refreshed three times a week. PC12 cells were treated with NGF (7S, 50 ng·mL−1; Invitrogen, Carlsbad, CA, USA) for 5 days prior to [3H]-noradrenaline uptake and release.
Determination of [3H]-noradrenaline release from detergent-permeabilized PC12 cells
The amount of secreted [3H]-noradrenaline was determined in digitonin-permeabilized cells as described previously [20,27]. In brief, PC12 cells were grown in 12-well plates at a density of 4 × 105 cells·dish−1. After the cells adhered to the plates (20–24 h), they were preincubated with peptides and [3H]-noradrenaline (1 μCi·mL−1) at 37 °C for 90 min in Krebs/Hepes solution (140 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 2.5 mm CaCl2, 1.2 mm MgSO4, 11 mm glucose and 15 mm Hepes/Tris, pH 7.4) containing 10 μm digitonin to permeabilize the cell. Cells were washed four times to remove the unincorporated radiolabeled compound. Cells were then depolarized with a high-K+ solution (115 mm NaCl, 50 mm KCl, 1.2 mm KH2PO4, 2.5 mm CaCl2, 1.2 mm MgSO4, 11 mm glucose and 15 mm Hepes/Tris, pH 7.4) for 15 min to assess the stimulated release. Extracellular media were transferred to scintillation vials and then measured by liquid scintillation counting. The amount of [3H]-noradrenaline release was calculated according to the following equation: amount of [3H]-noradrenaline release = (cpm of high K+-stimulated sample – cpm of basal level release) per mg of protein.
Cells were washed twice with ice-cold NaCl/Pi after treatment, and then lysed with RIPA buffer (Cell Signaling #9806) directly in the plate wells after the removal of media. Cell lysates were obtained by centrifugation at 13 000 g for 15 min at 4 °C. Protein concentrations were determined by the Bio-Rad protein assay kit using BSA as a standard. Equal amounts of protein (50 μg) from the cell lysates were dissolved in Laemmli’s sample buffer (not boiled), electrophoresed (SDS/PAGE) and transferred to a nitrocellulose membrane. The membranes were then blocked with NaCl/Pi/0.1% Tween 20 (NaCl/Pi-T buffer) containing 1% skim milk and 1% BSA for 1 h at room temperature. Thereafter, the membranes were incubated overnight at 4 °C with a 1 : 1000 dilution of anti-(SNAP-25) mAb. Membranes were washed three times with NaCl/Pi-T and further incubated with a 1 : 1000 dilution of horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Membranes were then washed extensively three more times with NaCl/Pi-T and developed using an enhanced chemiluminescence solution.
All experimental data were examined by analysis of variance (anova) procedures, and significant differences among the means from triplicate determinations were assessed using Duncan’s multiple range tests and the Statistical Analysis System, version 8.2 (SAS Institute Inc., Cary, NC, USA).
This study was supported by a Korea Research Foundation Grant (KRF-2004-C00197).