Marys Medicine

Pnas201006899 22145.22150

Dissection of SNARE-driven membrane fusion andneuroexocytosis by wedging small hydrophobicmolecules into the SNARE zipperYoosoo Yanga,1, Jae Yoon Shina,1, Jung-Mi Oha, Chang Hwa Junga, Yunha Hwanga, Sehyun Kima, Jun-Seob Kima,Kee-Jung Yoona, Ji-Young Ryub, Jaeil Shinc, Jae Sung Hwangd, Tae-Young Yoonb, Yeon-Kyun Shinc,e,2,and Dae-Hyuk Kweona,2 aSchool of Life Science and Biotechnology and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746, South Korea; bDepartment of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 760-749, South Korea; cDepartment of Biochemistry,Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011; dSkin Biotechnology Center, Graduate School of Biotechnology and Institute of LifeScience and Resources, Kyung Hee University, Yongin 446-701, Korea; and eIntegrative Biology and Biotechnology, Pohang University of Science andTechnology (POSTECH), Pohang 790-784, Korea Edited by Josep Rizo, University of Texas Southwestern Medical Center, Dallas, TX, and accepted by the Editorial Board November 8, 2010 (received for reviewMay 18, 2010) Neuronal SNARE proteins mediate neurotransmitter release at the allowed us to understand the basic architecture of the putative synapse by facilitating the fusion of vesicles to the presynaptic plasma membrane. Cognate v-SNAREs and t-SNAREs from thevesicle and the plasma membrane, respectively, zip up and bring about the apposition of two membranes attached at the C- SNARE-Driven Membrane Fusion Can Be Controlled by SHMs with terminal ends. Here, we demonstrate that SNARE zippering can Different Modes of Action. As an initial step to examine the feasi- be modulated in the midways by wedging with small hydrophobic bility of whether SHM works as a wedge for the SNARE zippering, molecules. Myricetin, which intercalated into the hydrophobic in- 39 polyphenolic compounds representing 12 subgroups were ner core near the middle of the SNARE complex, stopped SNARE screened for inhibitory activity against SNARE-driven proteoli- zippering in motion and accumulated the trans-complex, where posome fusion (11). We used polyphenolic compounds as a source the N-terminal region of v-SNARE VAMP2 is in the coiled coil with for SHM because they are abundant in nature and are known to be the frayed C-terminal region. Delphinidin and cyanidin inhibited versatile in helical bundle binding (12–15). The SHMs displayed N-terminal nucleation of SNARE zippering. Neuronal SNARE com- a wide range of inhibitory activity during the initial screen, which plex in PC12 cells showed the same pattern of vulnerability to was conducted at a concentration of 20 μM, equivalent to the small hydrophobic molecules. We propose that the half-zippedtrans concentration of t-SNARE proteins used in the fusion assay -SNARE complex is a crucial intermediate waiting for a calcium The nine most effective compounds were selected trigger that leads to fusion pore opening.
for subsequent experiments, and the least effective compound, polyphenol hemifusion neurotransmission neuron kaempferol, was used as a negative control. Next, the degree ofinhibition of lipid mixing (Fig. 1A) and SNARE complex forma- tion (Fig. 1B) were determined for each compound at a concen- eurotransmitter release at the synapse, which serves as the tration of 10 μM. The extent of SNARE complex formation was brain's major form of cell–cell communication, requires the assessed by exploiting the SDS-resistant property of the core fusion of synaptic vesicles with the presynaptic plasma membrane.
complex (16). For all 10 compounds tested, the degree of SNARE Soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor (SNARE) proteins mediate this synaptic fusion event complex formation inhibition correlated well with that of lipid- (1–5), and the formation of a four-helical bundle (6–8) is believed mixing inhibition (Fig. 1C), suggesting that SHM-mediated in- to generate the force required for fusion. A zipper model has hibition of membrane fusion is likely a direct consequence of in- been proposed for SNARE complex formation, initiating as- hibition of SNARE zippering. The tested compounds did not sembly at the N-terminal region and zipping toward the C-ter- cause liposome fusion without SNARE proteins and did not cause minal membrane-proximal region (6–9). To account for fast precipitation of proteoliposomes at the concentrations tested neuroexocytosis, the SNAREs in primed readily releasable vesicles ). We exclude the possibility that SHMs inhibited anti- have been proposed as being partially zipped in the trans-config- SNAP-25 antibody binding to SNARE complexes ( uration bridging the two membranes.
Thus, the results of the in vitro fusion assay indicate that certain Although the structure of the fully assembled cis-SNARE SHMs are capable of down-regulating SNARE complex forma- complex, which is believed to represent the postfusion state, has tion, thereby inhibiting membrane fusion in a concentration- been determined (10), the structure of the trans-complex is poorly dependent manner ( understood and is purely imaginary, most likely because of itsinherently transient nature. Precisely linking the degrees ofSNARE zippering to specific stages of membrane fusion seems to Author contributions: Y.Y., T.-Y.Y., Y.-K.S., and D.-H.K. designed research; Y.Y., J.Y.S., be prerequisite for determining the structure of the trans-complex J.-M.O., C.H.J., Y.H., S.K., J.-S.K., J.-Y.R., and J.S. performed research; J.Y.S., C.H.J., K.-J.Y.,J.-Y.R., and J.S.H. contributed new reagents/analytic tools; Y.Y., Y.-K.S., and D.-H.K.
and for providing answers to the questions of how fast fusion is analyzed data; and T.-Y.Y., Y.-K.S., and D.-H.K. wrote the paper.
controlled in neurons and how the trans-complexes set up the The authors declare no conflict of interest.
readily releasable vesicles with other regulatory proteins.
This article is a PNAS Direct Submission. J.R. is a guest editor invited by the Editorial Board.
Here, we show that certain small hydrophobic molecules 1Y.Y. and J.Y.S. contributed equally to this work.
(SHM) enable layer-by-layer control of SNARE zippering by 2To whom correspondence may be addressed. E-mail: or wedging into various points of the SNARE zipper. SNARE- mediated membrane fusion is dissected via this wedge-like action This article contains supporting information online at of SHMs. Analysis of the captured replication fork-like structure PNAS December 21, 2010 vol. 107 no. 51 22145–22150

ControlKaempferol Pre incubation control Pre incubation control K after preincubation M after preincubation K before preincubation M before preincubation Pre incubation control Pre incubation control D after preincubation C after preincubation D before preincubation C before preincubation SNARE complex formation (%) Down-regulation of SNARE-driven membrane fusion by SHMs. (A) Percentage maximum fluorescence intensity was plotted as a function of time in the presence or absence of SHMs. VpS, the soluble domain of VAMP2 lacking the transmembrane domain. SHMs were added at 10 μM concentration. (B)SNARE complex formation was assessed after the membrane-fusion assay by Western blotting using anti-SNAP25 antibody. (C) Correlation between thedegree of membrane fusion and the amount of SNARE complex formed in the presence or absence of SHMs. The concentration of SHM was 10 μM, and thedegree of fusion and complex formation were determined after an 80-min fusion initiation. Chemical structures of delphinidin, cyanidin, myricetin, andkaempferol are shown in the Inset. Concentration-dependent inhibition of fusion by these four SHMs is shown in (D) Soluble SNARE motifs form SDS-resistant complexes with SHMs in the inner layer. In the upper two gels, soluble fragments of SNARE proteins (each 500 μg/mL) were mixed with 10 μM SHMsand separated by SDS/PAGE. Gels were either stained with Coomassie blue (Coo) or electroblotted onto nitrocellulose membrane followed by incubation withnitroblue tetrazolium. + and − indicate the presence or absence of SHMs, respectively. In the lower two gels, the same experiment was performed withpreformed core complexes. Results indicate that SHMs bind to inner layer of the SNARE complex. K, kaempferol; M, myricetin; D, delphinidin; C, cyanidin. (E)Experimental procedures to determine fusion step-specific effects of SHMs on membrane fusion. (F) Fusion step-specific effects of SHMs on membrane fusion.
(G) Western blot analysis of SNARE complex formation. B, addition of SHMs before preincubation; A, addition of SHMs after preincubation.
We performed further experiments using the three most effi- a slight effect (Fig. 1F). In contrast, when the SHMs were in- cient compounds, delphinidin, cyanidin, and myricetin, as well as troduced after the preincubation step, the effects became di- the least effective, kaempferol (insets in Fig. 1C). First, by using vergent. Myricetin largely retained its inhibitory activity, whereas soluble SNARE motifs, SHMs were shown to intercalate into the delphinidin and cyanidin showed little inhibitory effect. The inner layer of the SNARE complex. SNARE complex-bound amount of SNARE complex formed correlated well with the SHMs were detected by nitroblue tetrazolium staining, which results of the lipid-mixing assay (Fig. 1G).
allows colorimetric detection of protein-bound polyphenols, onlywhen the SHMs were added before complex formation, indicating Binding Sites of SHMs in the SNARE Complex and the Effect of that the inhibitory SHMs intercalate into the inner layer of the Differential Binding on Hemifusion. We performed Ala-scanning core complex during helical bundle formation (Fig. 1D). The experiments to locate potential binding region of the three SHMs did not bind to binary t-SNARE complex regardless of SHMs in the SNARE zipper. All amino acids located at internal the presence or absence of the Habc domain and transmembrane a and d positions of the VAMP2 helix were mutated to alanine domain of syntaxin 1 one at a time, with the exception of native alanine residues.
To gain further insight into the mechanism by which SHMs P20A and P23A mutants were also prepared because the inhibit membrane fusion, the fusion step-specific effect on proline-rich region is known to have affinity to some polyphenols SNARE-dependent proteoliposome fusion was examined (17).
(18), and VAMP2 contains the proline-rich region before the Preincubation of t- and v-SNARE vesicle mixtures at 4 °C is known SNARE motif. The effect of each Ala mutation was assessed to enrich N-terminal, partially zipped complex and enhance with the in vitro fusion assay (Fig. 2A). With only a few excep- membrane fusion when the temperature is subsequently elevated tions, most Ala mutations hampered SNARE-mediated fusion to 37 °C. SHMs were added to the reaction either before or after by ∼50%, emphasizing the importance of the conserved core this preincubation step (Fig. 1E). SNARE-mediated fusion was residues for the fusion activity. The Ala mutants were then dramatically reduced when myricetin, delphinidin, or cyanidin was subjected to the lipid-mixing assay in the presence of an SHM.
added before the preincubation step, whereas kaempferol had only We reasoned that when a binding site for a particular SHM is

P2 P2 L3 T3 V39 A V42 A M4 N49 A V53 A R56 A L6 L6 L7 F7 L8 Y88 A K9
P2 P2 L3 T3 V39 A V42 A M4 N49 A V53 A R56 A L6 L6 L7 F7 L8 Y88 A K9
P2 P2 L3 T3 V39 A V42 A M4 N49 A V53 A R56 A L6 L6 L7 F7 L8 Y88 A K9
P2 P2 L3 T3 V39 A V42 A M46 A N49 A V53 A R56 A L6 L6 L7 F7 L8 Y88 A K9
000 2000 3000 4000 000 2000 3000 4000 Determination of SHM wedging points in the SNARE zipper and correlation of the degree of zippering with membrane-fusion stage. (A) The effect of alanine mutations on relative fusion efficiency. RFmo denotes the relative fusion efficiency of alanine mutants compared with wild-type VAMP2. RFmi is theratio between percentage maximum fluorescence intensities obtained in the presence or absence of inhibitors for the same mutant, where the inhibitors aremyricetin (M), delphinidin (D), and cyanidin (C) from left to right. The mutations with significantly higher values (blue bars) above RFwi (black bars) indicatethat those mutations are not inhibited by the SHM and thus represent the points that the SHM wedges into the SNARE zipper. The first black bar in each setdenotes RFwi for each SHM. Equations used to obtain relative fusion efficiency values are described in (B) Percentage hemifusion state was determinedthrough the inner-leaflet mixing assay in the absence or presence of SHMs. The actual time-dependent traces are shown in each SHM was used at10 μM in both studies.
mutated to Ala, the mutant would lose ability to bind the SHM, fluorescence-quenching assays were performed with Cy5-labeled thereby relieving inhibition by that SHM The relative VAMP2 mutants (Fig. 3). Five Cy5-labeled VAMP2 mutants fusion efficiency, defined as the ratio between the percentage were prepared: Q33C and T35C to probe N-terminal zippering, maximum fluorescence intensity (PMF) measured in the pres- L54C and R56C for middle region zippering, and D68C for ence of the drug to the PMF measured in the absence of the C-terminal zippering. FO/F, where FO and F represent fluores- drug, was plotted along the VAMP2 helix sequence for each cence intensity measured in the absence or presence of the fluo- SHM. Myricetin appears to have two binding sites: a weak rescence quencher acrylamide, respectively, increases if acrylamide binding site coordinated by hydrophobic residues between resi- is readily accessible to Cy5, indicating the labeled position is frayed.
dues 20 and 35 of VAMP2 and the corresponding t-SNARE Conversely, the slope is less affected by the quencher when the hydrophobic layers and a strong binding site coordinated by the Cy5-label is positioned in the coiled-coil region. In this regard, hydrophobic layers in the middle region (residues 49–63 of preincubation at 4 °C (Fig. 3, scheme A) seems to result in the VAMP2 and the corresponding t-SNARE residues). In contrast, replication fork-like SNARE zipper, where the N-terminal region delphinidin and cyanidin both had only one strong binding site at of VAMP2 is in the coiled-coil structure and the C-terminal region the N-terminal region between residues 20 and 42. These results remains frayed. After membrane fusion at 37 °C (Fig. 3, scheme B), were consistent with the results from the preincubation experi- all labeled VAMP2 residues appeared to participate in SNARE ments. It is noted that the proline-rich region also contributes to complex formation. By contrast, in the presence of myricetin SHM binding to the N terminus of SNARE complex, perhaps (Fig. 3, scheme D), the fluorescence-quenching pattern was similar through hydrophobic interaction (18). Although high-resolution to those of scheme A, indicating that SNARE zipper wedged by structures of SHM-containing SNARE complexes need to be myricetin has the frayed C terminus.
determined for better understanding of binding mechanisms,we speculate that the proline-rich region caps the N-terminal Dissection of SNARE-Dependent Membrane Fusion in PC12 Cells with SNARE bundle to enhance the SHM binding and SHMs. The next natural question is whether these observations are also true in live cells. First, we tested whether the SHMs could We hypothesized that it would be possible to link the degree of regulate SNARE complex formation in live cells. We prepared SNARE zippering to specific stages of membrane fusion by har- NGF-treated PC12 cells loaded with [3H]noradrenaline. Based nessing the unique properties of each SHM. We measured hem- on the in vitro results, we expected that the SHM binding sites in ifusion by using sodium dithionite that selectively kills fluorescent the SNARE complexes would only be exposed during recycling of dyes in outer leaflets (19). Myricetin inhibited inner-leaflet mixing SNARE proteins. To induce SNARE recycling, the cells were efficiently but allowed outer-leaflet mixing, stalling as much as pretreated with a high concentration of KCl followed by imme- 80% of the fusion outcome at the hemifusion state (Fig. 2B). In diate addition of SHMs. A high concentration of KCl is known to contrast, delphinidin and cyanidin had just a slight effect on the induce depolarization of PC12 cells leading to SNARE-mediated fraction of hemifusion (Fig. 2B and Together, our results membrane fusion and release of neurotransmitters (20).
raise the possibility that myricetin arrests membrane fusion at After high-K+ pretreatment (Fig. 4A, red box), neurotrans- the hemifusion state by wedging into the middle region of the mitter release was strongly inhibited by SHM exposure (Fig. 4B, SNARE zipper.
red bars). The three most competent compounds, delphinidin, cyanidin, and myricetin, showed 75–80% inhibition at 10 μM, Overall Fold of the Half-Zipped SNARE Complex. To determine comparable to the calcium channel blocker verapamil (21, 22) and the rough structure of SNARE zipper wedged by myricetin, only slightly lower than the Clostridium botulinum neurotoxin D PNAS December 21, 2010 vol. 107 no. 51 22147

V only (Q33C)
V only (T35C)
V (Q33C) + T
V (T35C) + T
V only (L54C)
V only (R56C)
V (L54C) + T
V (
V only (D68C)
V (D68C) + T
[Acrylamide, mM]
[Acrylamide, mM]
[Acrylamide, mM]
[Acrylamide, mM]
Fluorescence-quenching experiments for Cy5-labeled VAMP2 mutants. (A) Schematic presentation of fluorescence-quenching experiments to de- termine the structure of the SNARE zipper wedged by myricetin. (B) Stern–Volmer plots for the five Cy5-labeled VAMP2 mutants measured under variousconditions as indicated in schemes A–D. FO and F represent fluorescence intensity measured in the absence or presence of the fluorescence quencheracrylamide, respectively. Arrows indicate that the residues are engaged into the coiled-coil region when t-vesicle is present. A smaller difference in quenchingefficiency (indicated by short arrows) may result from partial coiling (schemes A and D) or because of a mixture of free VAMP2 and the half-zippered complex(scheme C). (C) Structures of SNARE zipper expected from the fluorescence-quenching experiments.
(BoNT/D). This high-K+ pretreatment experiment appeared binding sites are concealed inside the N-terminal bundle and the to mirror the in vitro preincubation experiment in which SHMs C-terminal stronger myricetin-binding site is not yet zipped.
were added before 4 °C preincubation (Fig. 1 E and F). Alter- natively, the in vitro results of adding SHMs after 4 °C pre-incubation were expected to correspond to those from the In the present study, it was shown that dynamic SNARE zip- neuronal cells without high-K+ pretreatment (Fig. 4A, blue box).
pering process could be stopped at the point of interest by Only myricetin showed a significant inhibitory effect on neuro- wedging an appropriate SHM into the zipper. Delphinidin andcyanidin wedge into the SNARE zipper at the far N terminus exocytosis (Fig. 4B, blue bars), which is consistent with our in vitro (∼P20–V42 of VAMP2), thereby preventing N-terminal nucle- observations that myricetin hampered C-terminal zippering by ation of SNARE complex formation (Myricetin stops wedging the half-zipped partial complex.
subsequent C-terminal zippering by wedging into the middle Membrane proteins were extracted from PC12 cells and ana- region of the SNARE zipper after the N-terminal half is zipped lyzed by Western blotting using an antibody against SNAP-25 (Fig.
By harnessing these unique characteristics of SHMs, 4C). Depolarization by high K+ indeed induced SDS-resistant we dissected SNARE-driven membrane fusion and tried to link SNARE complex formation (low K+ vs. high K+). In the presence the degree of SNARE zippering to the specific membrane- of SHMs, depolarization-induced SNARE complex formation was reduced, which is in agreement with the neurotransmitter release Preincubation at 4 °C is known to enhance membrane fusion results. Delphinidin and cyanidin effectively reduced SNARE at the subsequently elevated temperature. This preincubation complex formation only when the neuronal cells were pretreated step is thought to accumulate the N-terminal partial complexes with high-K+ solution (Fig. 4C, red box). In contrast, myricetin ), which undergo complete zippering in a synchronized effectively reduced SDS-resistant complex formation even without manner when the temperature is raised (11). When t-vesicles high-K+ pretreatment (Fig. 4C, blue box). Thus, the conformation were mixed with vesicles containing Cy5-labeled VAMP2 at 4 °C, of the trans-SNARE complex in neuronal cells is likely to be similar we could confirm the formation of the N-terminal partial com- to that observed in vitro, where the delphinidin- and cyanidin- plex with frayed C-terminal SNARE motifs (Fig. 3). The partial The effect of SHMs on neurotransmitter release and SNARE complex formation. (A) Experimental procedures. Red box, high-K+ pretreatment; blue box, no pretreatment. (B and C) After 15 min of main high-K+-induced depolarization, the amount of released neurotransmitters (B) and the amount of SDS-resistant SNARE complexes (C) were quantified in the supernatant and membrane fraction, respectively. α, β, and γ indicate the time of sampling formembrane fraction as shown in A. Reduced band intensity indicated by γ compared with β in the red-boxed gel shows that the pretreatment of high K+effectively dissembles preexisting SDS-resistant SNARE complexes in the cells. Red, high-K+ pretreatment group; blue, no pretreatment group. The effect ofSHMs on cell viability is shown in . IC50 values of SHMs on neurotransmitter release are shown in . Statistical significance of differenceswas evaluated by ANOVA (*P < 0.05).
complex, however, has never been captured at physiological Evidence indicates that the hemifusion state is a stable in- temperatures. The arrested zippering at physiological tempera- termediate of exocytosis in neuronal cells in vivo (28), and two ture by myricetin provides information about the trans-complex.
membranes may be hemifused before Ca2+ influx (29, 30). The Because the myricetin binding site is predicted to be coordinated similar responses to SHM treatments observed in both recon- by the hydrophobic residues of VAMP2 ∼N49–L63 and corre- stituted proteoliposomes and neuronal PC12 cells suggest that sponding t-SNARE residues (Fig. 2A), and the partial complex the halfway zippering-mediated hemifusion state (may formed by 4 °C preincubation is susceptible to myricetin (Fig. 1 be also true in the neuron. The hemifusion state likely progresses E–G), the results suggested that the region of VAMP2 after N49 toward the fusion pore when calcium triggers full SNARE zip- remains unzipped in the partial complex. Because this partial pering ), with the help of other regulatory proteins such complex is not susceptible to delphinidin or cyanidin, which bind as synaptotagmin and complexin (31). We note, however, thatthere are contradictory results against the hemifusion state (32), before V42, the SHMs-assisted assays suggest the possibility that and there is the possibility, although unlikely, that myricetin's the N-terminal region up to M46 forms a coiled coil during 4 °C interaction with the membrane is in part responsible for hemi- preincubation. Static fluorescence-quenching experiments (Fig.
3) also support that this halfway N-terminal zippering up to M46 BoNTs elicit neuron-specific flaccid paralysis by specifically with the frayed C-terminal half of VAMP2 is likely to represent cleaving neuronal SNARE proteins. After the realization of the the trans-complex ).
therapeutic potentials of these otherwise fatal toxins, the Food It is believed that SNARE complex formation starts at the and Drug Administration approved the controlled use of BoNT/ membrane-distal N-terminal region and zips toward the mem- A for the treatment of several hypersecretion-related neurolog- brane-proximal C-terminal region (6–9), thereby effectively forc- ical diseases, such as strabismus, blepharospam, hemifacial ing the apposition of the two membranes. Also, it is widely ac- spasm, and cervical dystonia (33). Recently, the cosmetic use of cepted that N-terminal assembly of SNARE proteins underlies these toxins for treating glabellar facial lines and axillary hy- vesicle priming, whereas assembly of the C-terminal end drives perhidrosis has gained immense popularity. Can small molecules, fusion. In this model, an intermediate state of the SNARE com- with more conventional drug-like properties, also regulate plex is associated with the primed vesicle state, and subsequent SNARE complex formation and consequent neuroexocytosis? C-terminal assembly, assisted by other proteins such as synapto- Promisingly, our results imply that SNARE-wedging small mol- tagmin and complexin, leads to membrane fusion and the re- ecules might serve as SNARE-specific drugs, which will definitely laxation of trans-SNAREpins into cis-SNARE complexes. Many provide a tractable avenue in treating a variety of human hy- of recent studies on complexin- and synaptotagmin-modulated persecretion diseases. These small molecules can serve as gen- fast neuroexocytosis presume this model to be true to account for eral chemical platforms from which unique and potent SNARE- their findings (3, 10, 23–27). However, our results raise another specific drugs can be derived.
possibility, where N-terminal half zippering, which had been Materials and Methods merely considered responsible for vesicle docking, might be suf- cient to induce hemifusion and is the minimal zippering degree for membrane hemifusion. Our results suggest that the zippering degree responsible for hemifusion could be only up to residue 46 sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) were obtained from Avanti Polar Lipids. RPMI medium 1640, PNAS December 21, 2010 vol. 107 no. 51 22149 penicillin-streptomycin, horse serum, and FBS were purchased from GIBCO/ ferred to scintillation vials and measured by liquid scintillation counting. The BRL. Recombinant BoNT/D light chain was purchased from List Biological quantity of released neurotransmitter was calculated according to the fol- Laboratories. All other chemicals, including polyphenolic compounds, were lowing equation: The quantity of released [3H]noradrenaline = (cpm of high- purchased from Sigma-Aldrich.
K+-stimulated sample − cpm of basal level release)/mg of protein. The amountof secreted neurotransmitters was determined without pretreatment of PC12 Cell Culture and Determination of [3H]Noradrenaline Release. PC12 detergents for permeabilization except for VpS and BoNT/D. Cell per- cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The PC12 meabilization for incorporation of VpS and BoNT/D (5 nM) was accomplished cells were plated onto poly-D-lysine–coated culture dishes and were main- with 10 μM digitonin in low-K+ solution (34). For high-K+ pretreatment groups, tained in RPMI medium 1640 containing 100 μg/mL streptomycin, 100 U/mL polyphenols were not added before the first high-K+ treatment but were penicillin, 2 mM L-glutamine, and 10% heat-inactivated FBS at 37 °C ina 5% CO before the second high-K+ treatment.
2 incubator. The cell cultures were split once a week, and the me- dium was refreshed three times a week. PC12 cells were treated withNGF (7S, 50 ng/mL, Invitrogen) for 5 d before uptake and release of [3H] Statistical Analysis. All experimental data were examined by ANOVA pro- cedures, and significant differences among the means from three to five After NGF-treated PC12 cells were grown in 12-well plates at a density of repeats were assessed with Duncan's multiple range tests and the Statistical 4 × 105 cells per dish, small-molecule compounds (10 μM) and [3H]noradren- Analysis System, version 8.2 (SAS Institute).
aline (1 μCi/mL) were applied for 10 min in low-K+ solution (140 mM NaCl, 4.7mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, and ACKNOWLEDGMENTS. This work was supported by the Basic Science Re- 15 mM Hepes-Tris, pH 7.4). The cells were washed four times to remove the search Program (2007-D00243 and 2010-0015035), by the Mid-Career Re- unincorporated radiolabeled neurotransmitters. The cells were then depo- searcher Program (2009-0058612), and by the World Class University Program larized with a high-K+ solution (115 mM NaCl, 50 mM KCl, 1.2 mM KH2PO4, through the National Research Foundation of Korea. This work was also 2.5 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, and 15 mM Hepes-Tris, pH 7.4) supported by Amorepacific Corporation and Small and Medium Business for 15 min to assess the stimulated release. Extracellular media was trans- 1. Söllner T, et al. (1993) SNAP receptors implicated in vesicle targeting and fusion.
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