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Vol. 18, Issue 12, 4957-4968, December 2007

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Synaptotagmin C2A Loop 2 Mediates Ca²⁺-dependent SNARE Interactions Essential for Ca²⁺-triggered Vesicle Exocytosis

K. L. Lynch^*,, R.R.L. Gerona^*,, E. C. Larsen^*,, R. F. Marcia^*,, J. C. Mitchell^*,, and T.F.J. Martin^*

Departments of *Biochemistry and Mathematics, University of Wisconsin, Madison, WI 53706

Submitted April 24, 2007; Revised September 21, 2007; Accepted September 26, 2007
Monitoring Editor: Patrick Brennwald

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synaptotagmins contain tandem C2 domains and function as Ca²⁺ sensors for vesicle exocytosis but the mechanism for coupling Ca²⁺ rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca²⁺-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin–1 that mediate Ca²⁺-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca²⁺-dependent synaptotagmin–1 binding to SNAREs without affecting Ca²⁺-dependent membrane binding. The mutants failed to confer Ca²⁺ regulation on SNARE-dependent liposome fusion and failed to restore Ca²⁺-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca²⁺-dependent SNARE binding by synaptotagmin is essential for Ca²⁺-triggered vesicle exocytosis and that Ca²⁺-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca²⁺-dependent SNARE and membrane binding occur simultaneously.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Neurotransmitter, neuropeptide, and peptide hormone secretion is mediated by the fusion of vesicles with the plasma membrane in a reaction catalyzed by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. The vesicle SNARE VAMP-2 (aka synaptobrevin) assembles into a four-helix bundle with the plasma membrane SNAREs, SNAP25 and syntaxin-1 (Sollner et al., 1993a; Hayashi et al., 1994; Fasshauer et al., 1997; Poirier et al., 1998; Sutton et al., 1998), to promote close phospholipid bilayer apposition and potentially drive membrane fusion (Weber et al., 1998).

Peptide and transmitter release by SNARE complex–dependent vesicle exocytosis is tightly regulated by cytoplasmic Ca²⁺ (Douglas and Rubin, 1961; Katz and Miledi, 1965). Synaptotagmin proteins possess the requisite Ca²⁺-binding properties to serve as Ca²⁺ sensors that regulate vesicle exocytosis (Brose et al., 1992; Chapman, 2002; Sudhof, 2002; Rizo et al., 2006). Consistent with a Ca²⁺-sensor role, synaptotagmin–1 is essential for fast synchronous Ca²⁺-triggered neurotransmitter release (Geppert et al., 1994; Littleton et al., 1994) and mutant alleles alter the Ca²⁺-dependence of neurotransmitter release (Fernandez-Chacon et al., 2001; Littleton et al., 2001; Rhee et al., 2005). Ca²⁺ regulation of vesicle fusion is fully abrogated in cells that lack vesicle-resident synaptotagmins (Lynch and Martin, 2007). The cytoplasmic domain of synaptotagmin–1 (C2AB) also confers Ca²⁺ stimulation on SNARE-dependent liposome fusion in vitro (Tucker et al., 2004). The precise mechanism by which synaptotagmins mediate the coupling of cytoplasmic Ca²⁺ rises in vivo to SNARE-dependent vesicle fusion is incompletely understood.

Synaptotagmin–1 is composed of N-terminal luminal and transmembrane domains and a cytoplasmic domain containing membrane-proximal (C2A) and membrane-distal (C2B) C2 domains connected by a short linker. Five aspartates plus other residues in loops 1 and 3 of the eight-stranded β-sandwich C2 domains ligate 3 and 2 Ca²⁺ ions in C2A and C2B, respectively (Sutton et al., 1995, 1999; Ubach et al., 1998; Fernandez-Chacon et al., 2001). The intrinsic affinity for Ca²⁺ is quite low (Fernandez-Chacon et al., 2002) but is markedly enhanced by the interaction of the C2 domains with bilayers containing acidic phospholipids such as phosphatidylserine (PS; Zhang et al., 1998). Interactions with PS complete the Ca²⁺-binding sites on loops 1 and 3, and adjacent hydrophobic residues at the tips of these loops insert into the bilayer (Zhang et al., 1998; Bai et al., 2002; Frazier et al., 2003). Ca²⁺-dependent synaptotagmin binding to PS is an important coupling element in Ca²⁺-triggered vesicle fusion. Mutation of C2A loop 3 R233 reduces the Ca²⁺ affinity for PS binding by synaptotagmin–1 and reduces the Ca²⁺-dependent probability of transmitter release (Fernandez-Chacon et al., 2001). Conversely, mutation of hydrophobic residues in the Ca²⁺-binding loops 1 and 3 to tryptophan enhances the Ca²⁺ affinity for PS binding and correspondingly increases the Ca²⁺-dependent probability of transmitter release (Rhee et al., 2005). The ability of synaptotagmin C2AB to confer Ca²⁺ stimulation on SNARE-dependent liposome fusion also depends upon the presence of PS in liposomes (Bhalla et al., 2005). These findings indicate that PS-binding by synaptotagmin–1 is essential for coupling Ca²⁺ increases to membrane fusion.

Synaptotagmins also exhibit Ca²⁺-independent and -dependent interactions with SNARE proteins in vitro. Synaptotagmin–1 is coisolated with SNARE complexes from brain detergent extracts in the absence of Ca²⁺ (Bennett et al., 1992; Sollner et al., 1993a; Schiavo et al., 1997). However, synaptotagmin–1 also engages in Ca²⁺-stimulated interactions with syntaxin-1 and SNAP25 (Chapman et al., 1995; Li et al., 1995; Kee and Scheller, 1996; Davis et al., 1999; Gerona et al., 2000) as well as with heterodimeric and heterotrimeric SNARE complexes (Davis et al., 1999; Gerona et al., 2000). Ca²⁺-dependent synaptotagmin-SNARE interactions provide an appealing mechanism for coupling Ca²⁺ rises to membrane fusion but the physiological role of this interaction has not been directly assessed. A major reason for this is the lack of information on regions in synaptotagmin that mediate SNARE interactions. Although NMR and crystal structures for synaptotagmin and SNARE complexes have been solved (Sutton et al., 1995, 1998, 1999; Shao et al., 1998; Fernandez-Chacon et al., 2001; Fernandez et al., 2001; Ernst and Brunger, 2003), structures of synaptotagmin-bound SNARE complexes are not yet available. Thus, precise point mutations in synaptotagmin that selectively affect SNARE binding without affecting PS binding have not been available to critically assess the functional role of Ca²⁺-dependent synaptotagmin-SNARE interactions.

In the current work, we identified sites on synaptotagmin–1 that bind SNAP25 using zero-length cross-linking. Mutation of sites in C2A and C2B was found to eliminate Ca²⁺-dependent synaptotagmin–1 binding to SNARE complexes without affecting PS- or phosphatidylinositol-4,5-bisphosphate (PIP₂)-containing membrane binding. The mutations eliminated the Ca²⁺ stimulation of SNARE-dependent liposome fusion by synaptotagmin–1 and prevented the restoration by synaptotagmin–1 of Ca²⁺-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. A structure-based model of C2A loop 2 interactions with the C-terminus of SNAP25 provided a novel view of simultaneous Ca²⁺-dependent SNARE and membrane binding that accounts for previous findings on synaptotagmin–1 function.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibodies and DNA Constructs
Antibodies used were synaptotagmin–1 monoclonal (clone 604.1; Synaptic Systems, Göttingen, Germany); SNAP25 monoclonal (Sternberger Monoclonals, Baltimore, MD); and CgB monoclonal (generously provided by W. Huttner, Max Plânck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). A synaptotagmin–1 polyclonal antibody was generated using a synaptotagmin–1 C2AB fusion protein. Plasmid pGEX-2T (Amersham Pharmacia Biotech, Piscataway, NJ) was used for Escherichia coli expression of recombinant proteins. The vectors encoding synaptotagmin–1 C2AB, C2A, and C2B, SNAP25B, and VAMP2 were kindly provided by R. H. Scheller (Genentech). Vectors encoding syntaxin 1A and synaptotagmin–1 were kindly provided by E. Chapman (University of Wisconsin). Glutathione S-transferase (GST) fusion proteins were produced in E. coli by standard methods and purified by glutathione-agarose chromatography (Amersham Pharmacia Biotech). Synaptotagmin–1 C2AB was purified on glutathione-Sepharose 4B using nuclease and high-salt washes to remove contaminants (Tucker et al., 2003). Plasmids for generating full-length syntaxin and SNAP25 heterodimer (pTW34) and full-length VAMP2 (pTW2) were generously provided by J. E. Rothman (Columbia University) and T. Weber (Columbia University). The plasmid encoding ANF-EGFP was kindly provided by E. Levitan (University of Pittsburgh School of Medicine, Pittsburgh, PA). Oligonucleotides encoding synaptotagmin–1/-9 short hairpin RNAs (shRNAs; Lynch and Martin, 2007) were ligated into pSHAG-1 vector (Paddison et al., 2002) via BamHI and BseRI sites. The open reading frame of synaptotagmin–1 was reverse-transcribed and PCR-amplified from rat PC12 cells and ligated into pcDNA3.1 (Invitrogen, Carlsbad, CA). The QuickChange Site-Directed Mutagenesis method (Stratagene, La Jolla, CA) was used to generate the synaptotagmin–1 silent mutant, pcDNA3-syt I sm (Lynch and Martin, 2007) and synaptotagmin–1 mutants in pGEX-2T-syt I C2AB and pcDNA3-syt I sm.

Cross-Linking and Mass Spectrometric Analysis
The SNAP25 protein used in binding and cross-linking studies exhibited 68% -helical content (estimated by circular dichroism), indicating that it was natively folded. Previous studies (Gerona et al., 2000) indicated that synaptotagmin bound SNAP25 and SNAP25/syntaxin heterodimers similarly. Cross-linking was performed in 1-ml reactions containing 20 mM HEPES (pH 7.2), 150 mM KCl, and 2 mM EGTA or 1 mM CaCl₂. Wild-type C2AB, 1 µM, or oligomerization-deficient (Chapman et al., 1998) C2AB K326A/K327A and 1 µM SNAP25 were incubated for 2 h at 4°C, followed by an additional 2-h incubation at 4°C in the presence of 2 mM 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 5 mM N-hydroxysulfosuccinimide (sulfo-NHS; Pierce Chemical, Rockford, IL). Cross-linking was quenched by the addition of 10 mM hydroxylamine. Protein was precipitated in 10% trichloroacetic acid and analyzed by SDS-PAGE for staining with colloidal Coomassie Blue. Protein bands were excised, washed and destained and gel fragments were reduced, alkylated, and digested with trypsin. Peptides were extracted and subjected to MALDI mass spectrometry at the University of Wisconsin-Madison Biotechnology Mass Spectrometry Facility. Peptide identity was determined using the MS Digest program (UCSF Protein Prospector). Results in Figure 1 are representative of three experiments, with similar results for C2AB or oligomerization-deficient C2AB K326A/K327A.

SNARE-containing Liposomes, Fusion Assay, and Binding Studies
All lipids were purchased from Avanti Polar Lipids (Alabaster, AL). Incorporation of VAMP-2 and SNAP25/syntaxin-1 into liposomes was conducted as described (Weber et al., 1998). VAMP-2 liposomes were formed by dialysis using a lipid mix comprised of 82% 1,2-dioleoyl-sn-glycero-phosphatidylcholine (DOPC), 15% 1,2-dioleoyl-sn-glycero-phosphatidylserine (DOPS), 1.5% N-(7-nitro-2–1,3-benzoxadizol-4-yl)-1,2-dipalmitoyl-sn-glycero-phosphatidylethanolamine (NBD-PE, donor), and 1.5% N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl-sn-glycero-phosphatidylethanolamine (Rh-PE, acceptor). SNAP25/syntaxin-1 liposomes were formed from 85% DOPS and 15% DOPC (mol/mol). VAMP-2 and SNAP25/syntaxin-1 liposomes contained 90 copies or 80 copies of proteins/liposome, respectively. Fusion assays were carried out as described (Weber et al., 1998; Tucker et al., 2004). t-SNARE liposomes, 45 µl, and 5 µl of NBD/Rhodamine-PE–labeled v-SNARE liposomes along with 10 µM C2AB or mutants in reconstitution buffer (10 mM HEPES, 150 mM KCl) were mixed in either 0.2 mM EGTA or the indicated concentration of Ca²⁺ in a total volume of 75 µl. NBD fluorescence was monitored for 2 h at 37°C. Dodecylmaltoside was added to a final concentration of 1% vol/vol, and NBD fluorescence was monitored for an additional 10 min. Data were plotted as % maximum NBD fluorescence and curve fitting was carried out using Prism 3.0 software with the variable slope sigmoidal dose–response function.

Flotation assays were carried out as described (Tucker et al., 2004) with modifications. Liposomes containing SNAP25 and syntaxin were mixed with 10 µM C2AB in either 0.2 mM EGTA or the indicated concentration of Ca²⁺ and subject to buoyant density flotation on Accudenz gradients (40, 30, and 0% vol/vol) by centrifugation at 45,000 rpm at 4°C for 4 h. Material at the 0%/30% interface was collected, analyzed by SDS gel electrophoresis and stained with colloidal Coomassie Blue. Coomassie-stained bands were quantified with a Molecular Dynamics SI Densitometer using Image Quant software (Sunnyvale, CA).

Binding studies for Figure 3a were conducted in 200 µl reactions containing 20 mM HEPES (pH 7.2), 150 mM KCl, 2 mM EGTA or 1 mM CaCl₂, 1% Triton X-100, and 0.5% cold fish skin gelatin (Sigma, St. Louis, MO). Ternary SNARE complexes were formed by overnight incubation at 4°C of equimolar amounts of SNAP25, VAMP, and GST-syntaxin. Glutathione-agarose beads containing 1 µM GST-SNARE complex were incubated with 1 µM C2AB. After a 2-h incubation at 4°C, beads were recovered by centrifugation, washed, eluted in sample buffer, and analyzed by SDS-PAGE and immunoblotting with synaptotagmin antibody using enhanced chemiluminescence with horseradish peroxidase–conjugated goat anti-rabbit IgG (Pierce Chemical). Western blots were quantified with a Molecular Dynamics SI Densitometer using Image Quant software.

Cell Culture, Transfection, Immunoblot Analysis, and Immunocytochemistry
PC12 cells were cultured in DMEM supplemented with 5% horse serum and 5% calf serum. Transfection was conducted by electroporation using an Electroporator II (Invitrogen). The synaptotagmin–1/9-null PC12 cell line was isolated as described previously (Lynch and Martin, 2007). Protein expression levels were determined from total cell lysates prepared in 1 mM phenylmethylsulfonyl fluoride and 1% Triton X-100 and clarified by centrifugation at 16000 x g for 5 min. Total protein, 20 µg, determined by bicinchoninic acid (BCA; Pierce Chemical) was loaded per lane for gel electrophoresis. Immunoblot analysis was conducted by standard methods.

For immunocytochemistry, cells were plated on poly-DL-lysine– and collagen-coated coverslips. Cells were washed with phosphate-buffered saline (PBS), fixed with 4% formaldehyde (wt/vol), permeabilized with 0.3% Triton X-100 in PBS, and blocked in 10% fetal bovine serum (FBS) in PBS. Primary and secondary antibodies were diluted in FBS blocking solution. Coverslips were mounted on slides with Mowiol 4–88 Reagent (Calbiochem, La Jolla, CA), and cells were imaged on a Nikon C1 laser scanning confocal microscope (Melville, NY) with a 60x oil immersion objective with NA 1.4. Z-series images were obtained with 250-nm sectioning with oversampling. The resulting Z-stacks were deconvolved using Autodeblur/autovisualize software (AutoQuant Imaging, Rochester, NY).

Exocytosis Assay
Cells were transiently transfected and plated on 35-mm glass-bottom dishes (MatTek, Ashland, MA) coated with poly-DL-lysine and collagen. After 48 h, cells were imaged on a Nikon total internal reflection fluorescence (TIRF) Microscope Evanescent Wave Imaging System on a TE2000-U Inverted Microscope (Nikon) and an Apo TIRF 100x, NA 1.45 (Nikon) objective lens. Enhanced green fluorescent protein (EGFP) fluorescence was excited with the 488-nm laser line of an argon ion laser. Cells were imaged in basal media (15 mM HEPES, pH 7.4, 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl₂, 0.5 mM MgCl₂, 5.6 mM glucose, 0.5 mM ascorbic acid, and 0.1% bovine serum albumin [BSA]) or depolarization medium (same as basal medium with 95 mM NaCl and 56 mM KCl). Images were acquired at 250-ms intervals with a CoolSNAP-ES Digital Monochrome CCD camera system (Photometrics, Woburn, MA) controlled by Metamorph software (Universal Imaging, West Chester, PA). All analysis was done using Metamorph software (Universal Imaging).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca²⁺ Stimulates the Formation of a Synaptotagmin-SNAP25 Cross-linked Product
Acidic residues in the C-terminus of SNAP25 are required for Ca²⁺-dependent synaptotagmin–1 binding and Ca²⁺-triggered vesicle exocytosis (Zhang et al., 2002). To determine the sites on both proteins that mediate synaptotagmin–1 interactions with SNAP25, we used cross-linking with EDC and sulfo-NHS. Synaptotagmin binding to SNARE proteins is electrostatic (Tang et al., 2006) and the zero-length cross-linker EDC converts carboxylate and amine groups in close proximity to amide bonds.

A cross-linked adduct containing the cytoplasmic domain of synaptotagmin–1 (termed C2AB) and SNAP25 was observed in both the absence and presence of Ca²⁺ (Figure 1a). The molecular weight of the C2AB-SNAP25 adduct (65 kDa) indicated that it consisted of one C2AB cross-linked to one SNAP25, as anticipated from the stoichiometry of binding (Davis et al., 1999; Gerona et al., 2000). The presence of Ca²⁺ markedly enhanced the yield of cross-linked C2AB-SNAP25 (Figure 1a) similar to the effect of Ca²⁺ in binding studies (Davis et al., 1999; Gerona et al., 2000). At the protein concentrations used (1 µM), we detected very little cross-linking of either C2A or C2B with SNAP25 in the absence or presence of Ca²⁺ (Figure 1b), which is similar to previous binding studies (Schiavo et al., 1997; Gerona et al., 2000; Tucker et al., 2003). These studies confirmed that both C2 domains in C2AB are required for optimal binding to SNAP25 and that this interaction is strongly stimulated by Ca²⁺.

View larger version (40K): [in this window] [in a new window]   Figure 1. Ca2+ stimulates the formation of a C2AB-SNAP25 cross-linked adduct. (a) Wild-type C2AB (not shown) or C2AB K326A/K327A (1 µM) and SNAP25 (1 µM) were incubated in the absence (–) or presence (+) of 1 mM CaCl2 before the addition of cross-linker (EDC/sulfo-NHS) and analyzed by SDS PAGE with Coomassie staining. C2AB K326A/K327A was used to minimize oligomerization (Chapman et al., 1998), but identical results were obtained with wild-type C2AB and no self cross-linking of C2AB was observed. Bands corresponding to SNAP25, C2AB, SNAP25 dimer, C2AB-SNAP25 adduct (*), and hsp70 are indicated. Lane on right was loaded with 10% input lacking cross-linker. (b) Both C2 domains are required for optimal cross-linking of C2AB to SNAP25. C2AB, C2A, or C2B fusion proteins (1 µM) were incubated with SNAP25 (1 µM) in the absence (–) or presence (+) of 1 mM Ca2+ before cross-linking. Bands corresponding to C2AB-SNAP25 (*), C2B-SNAP25, C2A-SNAP25, or free proteins are indicated.

View larger version (33K): [in this window] [in a new window]   Figure 2. Schematic summary of C2AB interactions with SNAP25. The presence of Ca2+ altered interactions between C2A and C2B domains with SNAP25. For C2A, residues 191-196 bound the C terminal portion of the N terminal helix of SNAP25 (residues 77-83) in the absence but not presence of Ca2+ (orange). In the presence of Ca2+, C2A residues 191–200 bound the C terminal helix of SNAP25 (residues 176-201; blue). For C2B, residues 301-313 bound to the N terminus of SNAP25 (residues 32-40; green) in the absence of Ca2+ but shifted to C2B residues 289-297 in the presence of Ca2+.

Two peaks with mass/charge ratios of 1523.8 and 2468.5 were observed only in the absence of Ca²⁺ (Table 1). The 1523.8 peak corresponded to synaptotagmin (191–196) cross-linked to SNAP25 (77–83). This cross-link represents an interaction between residues of C2A β4 with residues in the C-terminal portion of the N-terminal helix of SNAP25 (Figure 2). Combined with the preceding results, it was apparent that adjacent residues in the β4-loop 2 region of C2A shift their binding from C-terminal residues in the N-terminal helix (SN1) to C-terminal residues in the C-terminal helix (SN2) of SNAP25 when Ca²⁺ is bound (Figure 2). Thus, the β4-loop 2 region of C2A containing residues 191–200 operates as a Ca²⁺-dependent switch for SNAP25 interactions.

Interactions of synaptotagmin C2B with SNAP25 were detected in the absence of Ca²⁺ (Table 1). The 2468.5 peak corresponded to synaptotagmin (301–313) cross-linked to SNAP25 (32–40). The 2045.2 peak was observed in both the presence or absence of Ca²⁺ (Table 1) that corresponded, respectively, to synaptotagmin (289–297) cross-linked to SNAP25 (32–40). Thus, this loop 1 region of C2B encompassing residues 289–313 engaged the N-terminal portion of the N-terminal helix of SNAP25 (SN1) in interactions in the absence of Ca²⁺ (Figure 2). In the presence of Ca²⁺, the more N-terminal portion of the C2B loop 1 (residues 289-297) maintained SNAP25 binding, whereas SNAP25 binding by the C-terminal portion of the C2B loop 1 (residues 301-313) was not maintained in the presence of Ca²⁺ (Table 1). Because the C2B loop 1 contains Ca²⁺ ligands D303 and D309, Ca²⁺-binding may directly occlude SNAP25 interactions with this region. Thus, C2B exhibited more subtle Ca²⁺-dependent switching in its interactions with SNAP25 compared with that of C2A.

The R199A/K200A Mutation Reduces Ca²⁺-stimulated Synaptotagmin Binding to SNAREs
The Ca²⁺ switch region of C2A encompassing residues 191-200 that shifts interactions with SNAP25 upon Ca²⁺ binding consists of a positively charged region of C2A comprising β4-loop 2. To determine the importance of this C2A region for interactions with SNAP25, basic residues were mutated to alanine and effects on Ca²⁺-dependent SNARE binding were determined. C2AB harboring R199A/K200A mutations was found to exhibit decreased (by 70%) Ca²⁺-stimulated binding to immobilized ternary SNARE complexes (Figure 3a) and to SNAP25 but not to syntaxin (not shown). In contrast, Ca²⁺-dependent SNARE binding of C2AB with E194A/K196A and K191A/K192A mutations was indistinguishable from wild-type C2AB (Figure 3a). These findings further refined the cross-linking results and indicated that R199 and K200 of the C2A loop 2 region are essential for Ca²⁺-dependent synaptotagmin–1 binding. Significantly, the results also indicated that the same C2A residues important for Ca²⁺-dependent binding to the C-terminus of SNAP25 were essential determinants for Ca²⁺-dependent SNARE complex binding.

View larger version (28K): [in this window] [in a new window]   Figure 3. C2A loop 2 and C2B loop 1 mutations reduce Ca2+-stimulated binding of C2AB to SNARE complexes. (a) Wild-type C2AB, C2AB(R199A/K200A), C2AB (E194A/K196A) or C2AB(K191A/K192A), all at 1 µM, were incubated with 1 µM SNARE complex immobilized on glutathione-Sepharose beads in the absence (–) or presence (+) of 1 mM CaCl2. Binding of C2AB was determined by Western blotting after SDS PAGE. Mean values ± SE are shown (n = 3). (b) C2AB, C2AB(R199A/K200A) (RK) or C2AB (R199A/K200A/K297A/K301A) (RK/KK), all at 10 µM, were incubated with SNAP25/syntaxin-containing PC liposomes in the absence or presence of 1 mM Ca2+. Liposome-bound C2AB was isolated from Accudenz gradients and analyzed by SDS-PAGE and Coomassie Blue staining. (c) C2AB and indicated mutants were incubated with SNAP25/syntaxin-containing liposomes in the presence or absence of 1 mM Ca2+. RK corresponds to R199A/K200A. Bound C2AB was analyzed as in panel b, quantified by densitometry, and normalized to syntaxin in each gradient. Ca2+-dependent binding by C2AB mutants was expressed as the difference bound in the presence or absence of Ca2+ and normalized to binding by wild-type C2AB. Mean values ± SE are shown (n = 3). Differences in binding between wild-type C2AB and RK or K297A/K301A mutants, or between RK and RK/K297A/K301A mutants were significant (**p < 0.01; *p < 0.05).

SNARE-binding Mutations in C2AB Do Not Affect Ca²⁺-dependent PS or PIP₂ Interactions
Ca²⁺-dependent PS binding is the major biochemical activity of synaptotagmin–1 that has been implicated in Ca²⁺-coupling to membrane fusion (Geppert et al., 1994; Littleton et al., 1994; Zhang et al., 1998). The Ca²⁺-dependent SNARE-binding loop 2 of C2A is sufficiently distant from the PS-binding residues in loops 1 and 3 to allow for simultaneous PS and SNARE binding, which has been reported (Davis et al., 1999; but see Arac et al., 2003 for a different view). We directly assessed whether loop 2 residues were important for Ca²⁺-dependent PS-binding by conducting binding studies with PC/PS (85:15) liposomes. Wild-type C2AB exhibited Ca²⁺-dependent binding to PC/PS liposomes, but not to liposomes containing PC (not shown), with an EC₅₀ = 182 ± 21 µM Ca²⁺ (±SE, n = 3; Figure 4a). Indistinguishable Ca²⁺-dependent binding to PC/PS liposomes was observed for C2AB (R199A/K200A) as well as for C2AB (R199A/K200A/K297A/K301A), which exhibited EC₅₀ values of 155 ± 8 and 211 ± 34 µM Ca²⁺ (±SE, n = 3), respectively. Similarly, wild-type and mutant C2AB were found to exhibit indistinguishable Ca²⁺-dependent binding to PC/PIP₂ liposomes (Figure 4b). These results indicated that these residues in C2A loop 2 and C2B loop 1 are not required for Ca²⁺-dependent interactions of synaptotagmin–1 with phospholipid bilayers containing PS or PIP₂. In contrast, C2AB (R199A/K200A) as well as C2AB (R199A/K200A/K297A/K301A) mutants exhibited markedly impaired Ca²⁺-dependent binding to SNARE complexes embedded in PC-containing liposomes (Figure 4c). Thus, mutations in C2A and C2B that abrogated Ca²⁺-dependent SNARE binding did not affect Ca²⁺-dependent PS or PIP₂ binding. Synaptotagmin point mutants with this selectivity have not previously been generated and provide a unique opportunity to assess the functional role of Ca²⁺-dependent SNARE binding.

View larger version (16K): [in this window] [in a new window]   Figure 4. C2A loop 2 and C2B loop 1 mutants exhibit decreased Ca2+-dependent SNARE binding but normal Ca2+-dependent phosphatidylserine binding. (a) C2AB, C2AB(R199A/K200A) (RK) or C2AB(R199A/K200A/K297A/K301A) (RK/KK) were incubated with protein-free PC liposomes containing 15% PS at the indicated Ca2+ concentrations and bound C2AB was determined as in Figure 3b. Maximal bound C2AB was set equal to 100% and plotted (±SE, n = 3) as a function of [Ca2+]. (b) C2AB, C2AB(R199A/K200A) (RK), or C2AB(R199A/K200A/K297A/K301A) (RK/KK) were incubated with protein-free PC liposomes containing 5% PIP2 at the indicated Ca2+ concentrations and bound C2AB was determined as in Figure 3b. Fractional bound C2AB was plotted (±SE, n = 3) as a function of [Ca2+]. (c) C2AB and indicated mutants were incubated with PC liposomes containing SNARE complexes at the indicated Ca2+ concentrations, and bound C2AB was determined as in Figure 3b. The fraction of C2AB bound was plotted (±SE, n = 3) as a function of [Ca2+].

View larger version (29K): [in this window] [in a new window]   Figure 5. C2A loop 2 and C2B loop 1 mutations reduce Ca2+ stimulation of liposome fusion mediated by C2AB. (a) C2AB, 10 µM, stimulated the fusion of v-SNARE with t-SNARE liposomes in the presence of 100 µM Ca2+ but not in the presence of 0.2 mM EGTA. NBD fluorescence was expressed as percentage maximum fluorescence after 1% dodecyl maltoside addition. (b) C2AB, C2AB(K297A), or C2AB(K297A/K301A), all at 10 µM, were tested in the liposome fusion assay. C2AB(K297A/K301A) exhibited partial loss of function in conferring Ca2+ stimulation. (c) C2AB(R199A/K200A) (RK) or C2AB (R199A/K200A/K297A/K301A) (RK/KK) mutants failed to stimulate liposome fusion in the presence of 100 µM Ca2+. (d) C2AB, 10 µM, and mutants were incubated in the liposome fusion assay with indicated [Ca2+]. % maximal NBD fluorescence at 120 min was plotted as a function of [Ca2+] (±SE, n = 3). EC50 values for Ca2+ were 113 µM (±4 µM) for C2AB, 181 µM (±9 µM) for C2AB(R199A/K200A) (RK), and 187 µM (±9 µM) for C2AB (R199A/K200A/K297A/K301A) (RK/KK).

Synaptotagmin–1 C2A Loop 2 Mutations Fail to Mediate Ca²⁺-triggered Vesicle Exocytosis in PC12 Cells
The preceding results established that C2A (R199A/K200A) and C2B (K297A/K301A) mutations in synaptotagmin–1 selectively abolish Ca²⁺-dependent SNARE binding without affecting Ca²⁺-dependent PS or PIP₂ binding. This provided the opportunity to determine the role of Ca²⁺-dependent SNARE binding in the function of synaptotagmin–1 in cells. Synaptotagmins-1 and -9 mediate the Ca²⁺-triggering of vesicle exocytosis in PC12 cells. The concomitant shRNA-mediated down-regulation of both isoforms results in a complete block in Ca²⁺-triggered vesicle exocytosis at a step following vesicle docking (Lynch and Martin, 2007). Expression of either synaptotagmin with silent mutations that by-pass the shRNA targeting fully restores Ca²⁺-dependent vesicle exocytosis in the synaptotagmin–1/9-null PC12 cells (Lynch and Martin, 2007). Thus, we tested synaptotagmin–1 rescue constructs that encode C2A (R199A/K200A) or C2A (R199A/K200A) plus C2B (K297A/K301A) mutants. Wild-type and mutant synaptotagmins were expressed in the synaptotagmin–1/9-null PC12 cells at levels comparable (30–50%) to that in wild-type PC12 cells, which was proportional to transfection efficiency (Figure 6a). Expressed wild-type and mutant synaptotagmins were each correctly trafficked to dense-core vesicles as shown by colocalization with the dense-core vesicle content protein chromogranin B (Figure 6b). Thus, these mutations did not affect protein expression levels or proper targeting of the proteins to dense-core vesicles.

View larger version (23K): [in this window] [in a new window]   Figure 6. Synaptotagmin–1 mutants fail to restore Ca2+-dependent exocytosis in synaptotagmin-depleted PC12 cells. (a) Immunoblot analysis of wild-type PC12 cells, synaptotagmin–1/9-null cells, and null cells expressing wild-type synaptotagmin–1 or R199A/K200A (RK) or R199A/K200A/K297A/K301A (RK/KK) mutants. SNAP25 was used as a loading control. (b) Wild-type and synaptotagmin–1/9-null cells expressing synaptotagmin–1 or indicated mutants were fixed and stained with synaptotagmin–1 (green channel) and CgB (red channel) antibodies, and colocalization was determined (yellow). The synaptotagmin–1 mutants were localized to dense-core vesicles. (c) Cells expressing ANF-EGFP were stimulated with depolarization medium, and images were acquired at 0.25-s intervals. Fusion events were counted as a flash/puff of fluorescence for each cell type, and the sum of fusion events in 10-s intervals was determined. The sums of the average number of fusion events per cell over time (mean value ± SD) for wild-type (n = 25), synaptotagmin–1/9-null (n = 25), synaptotagmin–1/9-null + synaptotagmin–1 (n = 20), synaptotagmin–1/9-null + synaptotagmin–1 RK (n = 15), and synaptotagmin–1/9-null + synaptotagmin–1 RK/KK (n = 15) cells were plotted.

A Model for Ca²⁺-dependent Synaptotagmin C2A Function
Although NMR and crystal structures for synaptotagmin and SNARE complexes have been solved (Sutton et al., 1995, 1998, 1999; Shao et al., 1998; Fernandez et al., 2001), structures of synaptotagmin-bound SNARE complexes are not yet available. To predict the structure of a Ca²⁺-dependent complex between the C2A loop 2 region of synaptotagmin–1 and the C-terminus of SNAP25 in the SNARE complex, we used the ZDOCK program (Chen and Weng, 2002; Chen et al., 2003) and available structures of C2A and SNARE complexes.

An initial set of 40,000 binding complexes was generated by systematically docking the atomic coordinates of the SNARE complex to each of 20 NMR structures of the C2A domain. To predict complexes that represented the Ca²⁺-dependent interaction between C2A and SNAP25, we set restraints on the docking experiment. Our previous studies, confirmed by the cross-linking results, showed that three acidic residues in the C-terminus of SNAP25 (D179, D186, D193) are essential for Ca²⁺-dependent interactions between synaptotagmin–1 and SNARE complexes (Zhang et al., 2002). Fifty-nine docked complexes were identified in which C2A residues were within 5 Å of the three acidic residues of SNAP25. Complexes with C2A loop 2 residues K196, R199, and K200 in proximity to the three acidic residues in SNAP25 were found to be most prevalent (Figure 7a). We performed a complementary docking experiment in which C2A R199 and K200 residues were used as reference points to identify complexes with SNAP25 that were within 5 Å. Only 28 complexes were generated that satisfied this restraint, and three of these coincided with complexes from the first docking experiment. One of this set of three complexes is depicted in Figure 7b.

View larger version (51K): [in this window] [in a new window]   Figure 7. Ca2+-dependent C2A/SNARE binding interface derived from computational modeling. (a) Using ZDOCK, each of 20 NMR structures of C2A was computationally docked to the atomic coordinates of the SNARE complex crystal structure. The frequency of occurrence of individual C2A residues within 5 Å of acidic C-terminal SNAP25 residues D179, D186, and D193 is depicted. C2A is shown in a space filling model docked to a ribbon model of the SNARE complex. C2A residues are colored according to their frequency of proximity to SNAP25 D179, D186, or D193 from 0 (blue) to 20 (red) of 59. (b) The model shows the predicted low-energy docking orientation for Ca2+-dependent C2A-SNARE binding. The model corresponds to complex 1–1826 shown in Supplementary Tables S1 and S2. Close-up view depicts the binding interface for synaptotagmin–1 residues (K196, R199, and K200) with SNAP 25 residues (D179, D186, and D193).

The cluster of basic residues in C2A loop 2 (K196, R199, K200) is orthogonal to the membrane-penetrating face of C2A containing residues M173 and F234 in loops 1 and 3, respectively (Figure 8a). Thus, it was possible to model Ca²⁺-bound C2A in energetically favorable SNARE interactions while simultaneously penetrating the membrane (Figure 8b).

View larger version (79K): [in this window] [in a new window]   Figure 8. Membrane-docked model for Ca2+-dependent C2A-SNARE interaction. (a) Space-filling model of C2A depicting clustered basic residues of β4-loop 2 (K196, H198, R199, and K200) positioned orthogonally to membrane-inserting (M173 and F234) residues of loops 1 and 3. R233 is shown as positioned between both interfaces. (b) Model depicts simultaneous interactions of C2A with SNARE complex and membrane. Ca2+ ions (green spheres) bound by loops 1 and 3 with F234 penetrating the bilayer are shown. The orthogonal face containing K196, R199, and K200 interacting with SNAP25 C-terminal helix is depicted. R233 is shown between membrane- and SNARE-binding interfaces.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺-dependent interactions of synaptotagmin–1 with individual SNARE proteins and SNARE complexes in vitro have been extensively characterized because such interactions provide an attractive mechanism by which Ca²⁺ rises could be coupled directly to membrane fusion (Chapman et al., 1995; Li et al., 1995; Kee and Scheller, 1996; Davis et al., 1999; Gerona et al., 2000; Zhang et al., 2002). However, the physiological role of SNARE binding by synaptotagmin–1 had not been directly assessed because of the lack of precise point mutations that selectively eliminate Ca²⁺-dependent SNARE binding. Our cross-linking studies identified residues in the β4-loop 2 region of C2A that comprise a Ca²⁺-dependent switch for synaptotagmin–1 C2A interactions with SNAREs. In the presence of Ca²⁺, C2A loop 2 basic residues interact with acidic residues in the C-terminus of SNAP25, which is highly significant because this region of SNAP25 is essential for the Ca²⁺ regulation of vesicle exocytosis (Gerona et al., 2000; Zhang et al., 2002). The current work revealed that these C-terminal residues of SNAP25 comprise a direct site for Ca²⁺-dependent binding of synaptotagmin–1 to SNARE complexes.

The C2A loop 2 has not been previously implicated in the biochemical properties of synaptotagmin–1. An R199Q mutation was reported to have little effect on either PS or syntaxin binding (Shao et al., 1997; Zhang et al., 1998). The R199/K200 residues that mediate Ca²⁺-dependent SNARE binding reside at the apex of loop 2 in proximity to Ca²⁺-binding ligands on loops 1 and 3. R199 and K200 along with H198 and K196 form a positively charged cluster (see Figure 8a). Because Ca²⁺ binding does not cause a significant conformational change in C2A (Shao et al., 1996), it is thought that Ca²⁺-dependent effector binding by C2A is driven by nonspecific electrostatic interactions triggered by Ca²⁺ binding (Shao et al., 1997; Rizo and Sudhof, 1998). Ca²⁺ neutralizes acidic residues in loops 1 and 3 to shift the surface of C2A from a negative to positive electrostatic potential. This model has been successfully applied to understanding how Ca²⁺ binding drives the initial association of basic residues such as R233 (Zhang et al., 1998; Fernandez-Chacon et al., 2001) in C2A with negatively charged membranes (Murray and Honig, 2002). We propose that ligation of Ca²⁺ by loop 1 and 3 residues would shift the surface of C2A to a positive electrostatic potential that facilitates binding of R199 and K200 (and probably K196 and H198) to acidic residues in the C terminus of SNAP25. An electrostatic switch would enable rapid Ca²⁺-triggered binding kinetics as has been observed for synaptotagmin–1 interactions with SNAP25 in vitro (Bai et al., 2004).

Our model for synaptotagmin–1 interactions with SNAREs (Figure 8b) clarifies an important aspect of the Ca²⁺-regulated function of C2A by revealing that simultaneous interactions with anionic phospholipid membranes and SNARE complexes are feasible. Concomitant binding was demonstrated in one in vitro study (Davis et al., 1999) but not in another (Arac et al., 2003). In the absence of anionic phospholipids, three Ca²⁺ ions bind to C2A only at high [Ca²⁺] well beyond the intracellular physiological range (Ubach et al., 1998; Fernandez-Chacon et al., 2001). In the presence of anionic phospholipid membranes, affinities for Ca²⁺ binding to C2A increase up to 5000-fold (Zhang et al., 1998) bringing the effective [Ca²⁺] for binding into the intracellular physiological range. SNARE binding by synaptotagmin–1 can also be driven by Ca²⁺ in the absence of phospholipids but the affinities for Ca²⁺ are very low (100 µM) (Figure 4b), which contrasts sharply with Ca²⁺ concentrations (1–10 µM) that promote synaptotagmin–1 cross-linking to SNAP25 and trigger vesicle exocytosis in PC12 cells (Zhang et al., 2002). Ca²⁺-dependent SNARE binding by synaptotagmin–1 would likely not be achieved at intracellular Ca²⁺ concentrations in the absence of accompanying phospholipid binding. As our model shows (Figure 8a), the basic cluster of C2A loop 2 residues (K196, H198, R199, K200) is positioned orthogonally to loop 1 and loop 3 Ca²⁺-binding and membrane-inserting residues, which would allow simultaneous interactions with the SNAP25 C terminus in the SNARE complex and with the membrane. R233 is positioned at both SNAP25- and membrane-binding interfaces (Figure 8b), which accounts for the finding that C2A R233Q mutations decrease both Ca²⁺-dependent anionic membrane as well as SNAP25 interactions but not those with syntaxin (Fernandez-Chacon et al., 2001; Wang et al., 2003).

Cross-linking revealed that the C2B domain of synaptotagmin–1 also contributed to SNAP25 binding in the presence of Ca²⁺. We found that C2B loop 1 mutations partially reduced Ca²⁺-dependent binding of synaptotagmin–1 to SNARE complexes but fully (> 95%) abolished binding when combined with C2A loop 2 mutations. These studies revealed a requirement for both C2A and C2B domain residues in Ca²⁺-dependent interactions with SNAREs, which is consistent with previous findings that neither C2A nor C2B fragments possess full Ca²⁺-dependent SNARE-binding activity (Davis et al., 1999; Gerona et al., 2000), that Ca²⁺ ligand mutations in both C2 domains are needed to eliminate Ca²⁺-dependent SNARE binding (Earles et al., 2001) and that lengthening the linker between C2 domains reduces Ca²⁺-dependent SNARE binding (Bai et al., 2004). Unfortunately because of the limited information available, we were unable to model C2B interactions with SNAREs.

In vitro binding studies with C2A loop 2 and C2B loop 1 mutants revealed that these mutations result in a selective loss of Ca²⁺-dependent SNARE binding without affecting Ca²⁺-dependent PS or PIP₂ binding. No other defined point mutants in synaptotagmin–1 with these selective properties have been described although synaptotagmin–1 with inter-C2 domain linker extensions exhibit similar but attenuated properties (Bai et al., 2004). The selective loss of Ca²⁺-dependent SNARE binding by the C2A and C2B mutants provided the opportunity to critically assess this property of synaptotagmin–1 for its function as a Ca²⁺ sensor in regulated vesicle exocytosis. We found that the synaptotagmin–1 C2A (R199A/K200A) mutant was strongly impaired in restoring Ca²⁺-dependent exocytosis in synaptotagmin–1/9-null PC12 cells and that the C2A (R199A/K200A) C2B (K297A/K301A) double mutant was completely nonfunctional. Because both mutants were normally expressed and properly targeted to dense-core vesicles, these results provide unambiguous evidence for the essential role of Ca²⁺-dependent SNARE binding for synaptotagmin–1 function in regulated exocytosis. Moreover, they indicate that Ca²⁺-dependent interactions of synaptotagmin–1 with membranes are insufficient in the absence of SNARE binding to trigger fusion. The results highlight important roles for both C2 domains in function, which is consistent with previous genetic studies of synaptotagmin–1 in Ca²⁺-triggered vesicle exocytosis (Yoshihara et al., 2003). Particularly relevant is the observation that a C2A Ca²⁺ ligand mutation D232N increases the Ca²⁺ sensitivity of neurotransmitter release and the Ca²⁺-dependent binding of synaptotagmin–1 to SNAREs (Stevens and Sullivan, 2003; Pang et al., 2006), which is in accord with our conclusion that C2A contains a Ca²⁺-dependent switch for SNARE binding.

Although the current work provides important new insights on SNARE regulation by synaptotagmin–1 in Ca²⁺-triggered vesicle exocytosis, the precise role of Ca²⁺-dependent SNARE interactions remains to be clarified. Synaptotagmin–1 may function at an early stage to recruit SNAP25 into SNARE complexes for assembly of fusogenic SNARE complexes (Bhalla et al., 2006; Chen et al., 2001). Alternatively, a later-stage role for Ca²⁺-dependent synaptotagmin–1 binding to SNAP25 may be to displace SNARE complex-bound complexin, which could drive SNARE complexes into executing fusion (Reim et al., 2001; Tokumaru et al., 2001; Chen et al., 2002; Giraudo et al., 2006; Schaub et al., 2006; Tang et al., 2006). The Ca²⁺-dependent binding of C2A loop 2 of synaptotagmin–1 to C-terminal SNAP25-containing complexes may be well positioned to execute complexin displacement for triggering fusion.

In summary, the current work identified surfaces on the C2A and C2B domains of synaptotagmin–1 that mediate Ca²⁺-dependent binding to SNAP25 in SNARE complexes. Mutation of basic residues on these surfaces abrogate Ca²⁺-dependent interactions with SNARE complexes and abrogate Ca²⁺-triggered vesicle exocytosis without affecting Ca²⁺-dependent membrane binding. The results provide strong evidence that 1) Ca²⁺-dependent membrane interactions alone are insufficient for mediating the function of synaptotagmin–1 and 2) that the Ca²⁺ sensing role of synaptotagmin–1 in regulating vesicle exocytosis requires its direct interaction with the SNARE fusion machinery.

	ACKNOWLEDGMENTS

 
The authors thank Adam Steinberg for creating the model figures. This work was supported by US Public Health Service Grant DK25861 to T.F.J.M., by a Ruth L. Kirschstein predoctoral fellowship to K.L.L., by Department of Energy- Genomics to Life Grant DE-FG02-04ER25627 to J.C.M., and by support of R.F.M. by National Library of Medicine Grant 5T15LM007359-03.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-04-0368) on October 3, 2007.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

These authors contributed equally to this work.

Address correspondence to: T.F.J. Martin (tfmartin{at}wisc.edu).

Abbreviations used: SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SNAP25, synaptosomal associated protein of 25 kDa; VAMP, vesicle-associated membrane protein; PS, phosphatidylserine; PIP2, phosphatidylinositol-4,5-bisphosphate; TIRF, total internal reflection fluorescence; ANF-EGFP, atrial naturetic factor-enhanced green fluorescent protein; CgB, chromogranin B.

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