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Vol. 17, Issue 12, 5211-5226, December 2006

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C₂B Polylysine Motif of Synaptotagmin Facilitates a Ca²⁺-independent Stage of Synaptic Vesicle Priming In Vivo

Carin A. Loewen^*, Soo-Min Lee, Yeon-Kyun Shin, and Noreen E. Reist^*

*Molecular, Cellular, and Integrative Neuroscience Program, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523; and Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011

Submitted July 24, 2006; Revised September 6, 2006; Accepted September 7, 2006
Monitoring Editor: Jeffrey Brodsky

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca²⁺ affinity of release, and, at low Ca²⁺, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca²⁺ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt^null mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca²⁺-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca²⁺-independent docking and/or priming of synaptic vesicles in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synaptotagmin I is a synaptic vesicle protein whose cytoplasmic region contains two C₂ domains, C₂A and C₂B (Perin et al., 1991; Brose et al., 1992). Although synaptotagmin is a Ca²⁺ sensor for synaptic vesicle exocytosis (Brose et al., 1992; Geppert et al., 1994), primarily via its C₂B Ca²⁺-binding motif (Figure 1A, circles; Mackler et al., 2002; Nishiki and Augustine, 2004), it likely mediates other molecular interactions via additional functional motifs.

View larger version (51K): [in this window] [in a new window]   Figure 1. Synaptotagmin C2B polylysine region is highly conserved throughout evolution and is required for efficient synaptic transmission. (A) ClustalW sequence alignment of 56 highly conserved amino acids from the C2B domain of synaptotagmin. Bars indicate -sheets within the C2B domain. Circles mark the Ca2+-binding motif. Dotted line denotes the polybasic region. Asterisks mark the polylysine motif. (B) Representative traces showing evoked release during 0.05-Hz stimulation in HL3 saline containing 1.5 mM Ca2+ in transgenic controls (–/–;P[sytWT]) and polylysine motif mutants (–/–;P[sytKQ]). Each trace is an average of 10–12 individual traces from a single muscle fiber. Stimulus artifact has been removed for clarity. (C) The mean EJP amplitude in saline containing 1.5 mM Ca2+ was 14.2 ± 0.82 mV (mean ± SEM; n = 34 fibers) for polylysine motif mutants and 32.4 ± 0.99 mV (mean ± SEM; n = 42 fibers) for transgenic wild-type controls (p << 0.001).

Functional studies demonstrate that synaptotagmin I is required for synaptic vesicle endocytosis (von Poser et al., 2000; Jarousse and Kelly, 2001; Jarousse et al., 2003; Poskanzer et al., 2003; Llinás et al., 2004; Nicholson-Tomishima and Ryan, 2004), and in vitro studies suggest that the C₂B domain of synaptotagmin may mediate this role (von Poser et al., 2000; Jarousse and Kelly, 2001; Littleton et al., 2001; Jarousse et al., 2003; Llinás et al., 2004). Specifically, synaptic vesicle endocytosis may be mediated by the polylysine motif in the C₂B domain of synaptotagmin (Takei and Haucke, 2001; but see Poskanzer et al., 2006) via Ca²⁺-independent interactions with the clathrin adaptor protein AP-2 (Zhang et al., 1994; Chapman et al., 1998; Haucke and De Camilli, 1999; Haucke et al., 2000; Littleton et al., 2001; Grass et al., 2004) and/or with phosphatidylinositol bisphosphate (PIP₂) (Bai et al., 2004). Both AP-2 and PIP₂ play a role in clathrin-mediated endocytosis (Cremona and De Camilli, 2001; Hurley and Wendland, 2002).

A second proposed function for the polylysine motif of synaptotagmin is regulation of Ca²⁺-dependent processes (Chapman et al., 1998; Wu et al., 2003; Borden et al., 2005; Araç et al., 2006; Li et al., 2006). Polylysine motif mutations decrease the apparent Ca²⁺ affinity of synaptotagmin (Borden et al., 2005; Li et al., 2006). Furthermore, in vitro studies have implicated this motif in three Ca²⁺-dependent processes: the ability of synaptotagmin to oligomerize (Chapman et al., 1998; Wu et al., 2003), the ability of synaptotagmin to simultaneously bind to two membranes (Araç et al., 2006), and the ability of synaptotagmin to bind to negatively charged phospholipids (Li et al., 2006). The Ca²⁺-dependent oligomerization and phospholipid binding of synaptotagmin have been proposed to regulate the opening, dilation, or stability of the fusion pore (Wang et al., 2001; Bai and Chapman, 2004; Li et al., 2006). The simultaneous binding of synaptotagmin to two membranes has been proposed to accelerate membrane fusion by bringing the synaptic vesicle and plasma membrane into proximity (Araç et al., 2006).

Finally, the polylysine motif may participate in a Ca²⁺-independent interaction that either holds synaptic vesicles at release sites (docking) and/or subsequently increases their probability of release (priming). Priming likely consists of multiple maturation steps that transform vesicles from a docked but not releasable state, through multiple releasable states with differing release properties (Martin, 2003). In the absence of Ca²⁺, the polylysine motif of synaptotagmin preferentially binds to PIP₂-containing membranes, potentially mediating vesicle docking via a trans-interaction between the synaptic vesicle and the plasma membrane (Bai et al., 2004; Li et al., 2006) where PIP₂ is predominantly located (Holz et al., 2000; Micheva et al., 2001). This interaction could also mediate Ca²⁺-independent vesicle priming, because it increases the rate of synaptotagmin penetration into lipid membranes in vitro upon Ca²⁺ influx (Bai et al., 2004), which is postulated to be important for fusion (Bai et al., 2000, 2002; Wang et al., 2003; Rhee et al., 2005). Additionally, the polylysine motif of synaptotagmin binds to target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) heterodimers (syntaxin/synaptosome-associated protein 25 kDa [SNAP-25]) in the absence of Ca²⁺ (Rickman et al., 2004, 2006). This Ca²⁺-independent interaction may prime vesicles by bringing the synaptic vesicle (v)-SNARE (vesicle-associated membrane protein [VAMP] or synaptobrevin) into proximity with the t-SNARE heterodimer, preparing the vesicle for Ca²⁺-triggered fusion via formation or stabilization of the SNARE complex (VAMP/syntaxin/SNAP-25; Xu et al., 1999; Rickman et al., 2004, 2006; Bhalla et al., 2006). Indeed, inclusion of synaptotagmin in v-SNARE–containing vesicles directly accelerates SNARE-mediated membrane fusion in a Ca²⁺-independent manner (Mahal et al., 2002).

Using Drosophila mutants, we show that the polylysine motif plays a role during synaptic vesicle recycling after endocytosis and before fusion. This role serves to increase the rate of recovery from synaptic depression and increase the efficacy of synaptic vesicle fusion in vivo. Using a fluorescent lipid mixing assay, we show that the polylysine motif mediates the Ca²⁺-independent ability of synaptotagmin to accelerate SNARE-mediated fusion. We discuss these results in the context of a single deficit hypothesis where the polylysine motif plays an important role in Ca²⁺-independent docking/priming of synaptic vesicles.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fly Stocks
The polylysine motif (Figure 1A, asterisks) of Drosophila synaptotagmin was mutated (K_379,380,384Q), and the mutant synaptotagmin was expressed from a transgene in a synaptotagmin null (syt^AD4) line. The experimental flies (–/–;P[syt^KQ]) had the genotype yw; syt^AD4 P[elav-Gal4]/syt^AD4;P[UAS syt^{K379,380,384Q}]/+ and were generated as described previously (Mackler and Reist, 2001). The control flies (–/–;P[syt^WT]) expressed wild-type synaptotagmin I from a transgene (Mackler and Reist, 2001) and had the genotype yw; syt^AD4 P[elav-Gal4]/syt^AD4;P[UAS syt^WT]/+. Two independent transgenic wild-type synaptotagmin lines and two independent transgenic C₂B polylysine motif lines were generated. Because both lines of a given genotype had similar levels of evoked release (control lines [our unpublished data]; mutant lines, Mackler and Reist, 2001), one control line (2C) and one mutant line (#59) were used in subsequent experiments.

Solutions
Standard saline for these experiments is composed of 5 mM KCl, 2 mM CaCl₂, 130 mM NaCl, 2 mM MgCl₂, 36 mM sucrose, and 5 mM HEPES, pH 7.3 (Jan and Jan, 1976). In Ca²⁺-free saline, the CaCl₂ was replaced by additional MgCl₂. Stimulating saline contained 90 mM KCl, 5 mM CaCl₂, 45 mM NaCl, 2 mM MgCl₂, 36 mM sucrose, and 5 mM HEPES, pH 7.3. HL3 saline contained 5 mM KCl, 70 mM NaCl, 20 mM MgCl₂, 10 mM NaHCO₃, 5 mM HEPES, 115 mM sucrose, 5 mM trehalose, and 1.5 mM CaCl₂ (Stewart et al., 1994). For the Ca²⁺ dependence curves, the CaCl₂ concentration in the HL3 saline varied (0.3, 0.5, 0.6, 0.8, 1.0, 1.5, 2.5, 3.5, 5.0, or 6.0 mM), whereas the MgCl₂ concentration was held constant at 20 mM. Hyperosmotic saline consisted of HL3 saline containing 0.8 mM Ca²⁺ and 0.5 M sucrose.

Dye Uptake Assay
Synaptic boutons in Drosophila third instars were labeled with the activity-dependent, fluorescent dye FM 1-43 (Invitrogen, Carlsbad, CA). Larvae were dissected in Ca²⁺-free saline, stimulated for 6 min with stimulating saline, and then the stimulating saline was replaced with standard saline containing 3 µM tetrodotoxin (TTX) for a variable length of time (t = 0, 30, 60, or 180 s). TTX was added to block spontaneous activity coming from the attached CNS. After t, the preparation was incubated in standard saline + 3 µM TTX + 4 µm FM 1-43 dye for 6 min to load endocytosing vesicles, and then it was washed for 15 min with three changes of Ca²⁺-free saline to remove excess extracellular dye.

Fluorescence Microscopy and Image Processing
FM 1-43–labeled preparations were viewed using a 40x/0.80 numerical aperture water immersion objective lens (Leica, Bannockburn, IL) on a DMRA light microscope (Leica, Nussloch, Germany) equipped with epifluorescence optics (51019 EGFP/DsRed filter; Chroma Technology, Brattleboro, VT) and fitted with a microstepping servomotor in the z-axis. Images were captured with a Hamamatsu charge-coupled device camera (C4742–95). A through focal series by using 0.4-µm steps was taken of each labeled neuromuscular junction. All imaging was accomplished within 2 min of first exposure to light. Images were acquired, stored, and processed using Open Lab 2.2.0 software (Improvision, Boston, MA) on a Mac G-4 platform.

The average fluorescence intensity of 12–15 brightly stained boutons with diameters 2 µm were measured at each neuromuscular junction. Only boutons on muscle fibers 6 and 7 from segment 3 or 4 were used. Only one neuromuscular junction per preparation was imaged. Nine neuromuscular junctions (from 9 larvae) were analyzed for each genotype. Each bouton was imaged in its optimal focal plane. Images were imported into the public domain Object Image program (http://simon.bio.uva.nl/object-image.html). Background fluorescence was measured over nearby muscle nuclei and was subtracted from the fluorescence intensity of each bouton. Statistical comparisons between genotypes were tested with a mixed model analysis of variance (ANOVA) by using SAS software 6.12 (SAS Institute, Cary, NC).

Electrophysiology
Electrophysiology experiments were performed at room temperature in HL3 saline (Stewart et al., 1994) containing variable levels of Ca²⁺ (see above). Recordings were from muscle fiber 6 from abdominal segments 3 and 4 of third instar fillet preparations. Fibers were impaled with 15- to 45-M electrodes filled with a solution of 3 parts 2 M potassium citrate to 1 part 3 M potassium chloride or a 4 M potassium acetate solution. Segmental nerves were stimulated with a 5- to 10-µm diameter glass micropipette filled with HL3 to evoke excitatory junctional potentials. Nerves were stimulated with 1-ms pulses at the indicated frequency, and voltage traces were collected using an AxoClamp 2B (Molecular Devices, Sunnyvale, CA) and digitized using a MacLab4s A/D converter (Chart or Scope software; ADInstruments, Colorado Springs, CO). The resting membrane potential of each muscle fiber was normally between –65 and –45 mV but was maintained at about –55 mV by passing a bias current. KaleidaGraph software (Synergy Software, Reading, PA) was used to fit linear regression lines to the data in Figure 3B and the Hill equation [(Max x [Ca²⁺]ⁿ)/(EC₅₀ⁿ + [Ca²⁺]ⁿ)] to the data in Figure 3, A and C.

Hyperosmotic saline was pressure injected with a PLI-100 PICO-INJECTOR (Medical Systems, Greenvale, NY) onto the neuromuscular junction for 10 s with an injection pressure of 1.0 psi by using a glass micropipette with a tip diameter <1 µm. The recording electrode impaled the muscle at the extreme posterior end of the muscle, outside the area of hyperosmotic saline application, to reduce nonspecific leak current (Aravamudan et al., 1999). Voltage traces were collected as described above. In a blind analysis, the number of quantal events within 1-s intervals was binned during the 10-s hyperosmotic puff as well as during the 10 s before and after the puff.

Immunohistochemistry
Experimental and control third instars were dissected in the same dish in cold Ca²⁺-free HL3 saline; fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS); rinsed in PBS containing 0.1% Triton X-100 and 0.02% azide (PBST); and incubated overnight at 4°C in monoclonal antibody (mAb) nc82 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), diluted 1:100 in PBST containing 5% normal goat serum (PBST-NGS) and anti-GluRIII antibody (Marrus et al., 2004), diluted 1:200 in PBST-NGS. The preparations were washed in PBST, incubated for 1 h in Alexa Fluor 488 goat anti-mouse IgG (Invitrogen), diluted 1:500 in PBST-NGS and Texas Red goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:200 in PBST-NGS. Preparations were washed in PBST and mounted in Citifluor AF-1 (Ted Pella, Redding, CA).

Neuromuscular junctions from abdominal segments 3 or 4 were imaged on a Zeiss LSM 510Meta confocal microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with argon and HeNe1 lasers. Emissions were collected using a band pass 505–530 emission filter (for Alexa Fluor 488) or a long pass 585 emission filter (for Texas Red). Images were collected at 40 and 63x (pinhole set for 1 Airy unit for each), and confocal settings were adjusted to optimize the intensity range of the signal. For all images used for quantitative analysis, the settings were held constant between control and experimental preparations. Eight-bit images were acquired and z-stacks were converted to projections using Zeiss LM510 software.

For analysis, a region of interest (ROI) of constant area was drawn around each synaptic arborization as well as around a nearby area that contained only background staining. The histogram function of the software was used to determine the absolute number of pixels at every intensity (0–255) in these ROIs. To determine the intensity range of the signal (i.e., the range where the number of pixels in the signal was consistently greater than the number of pixels in the background), the number of pixels in the background ROI was subtracted from the number in the ROI containing the stained arborization for each intensity level. Once the intensity range of the signal was determined, the area of the signal was calculated by summing the number of pixels in the signal range. For Figure 2, the gamma setting of a Codonics NP-1600 Photographic Network Printer was adjusted to match the image appearance on the screen.

View larger version (62K): [in this window] [in a new window]   Figure 2. GluRIII and nc82 antibody staining was unchanged in polylysine motif mutants. Representative 40 and 63x images of neuromuscular junctions from transgenic controls (–/–;P[sytWT], top) and polylysine motif mutants (–/–;P[sytKQ], bottom). Junctions were stained with an anti-GluRIII antibody (red) to show postsynaptic receptors, and they were stained with mAb nc82 (green) to show active zones. Bar, 10 µm.

Plasmid Construction and Site-directed Mutation for Fusion Proteins
Syntaxin 1A (amino acids 4-288), SNAP-25 (amino acids 1-206), and VAMP2 (amino acids 1-116) from rat were all inserted into pGEX-KG (between EcoRI and HindIII sites) as glutathione S-transferase (GST) fusion proteins. Four native cysteines in SNAP-25, one in VAMP2 and three in syntaxin 1A, were replaced with alanines. The plasmid for rat synaptotagmin I with a truncated luminal domain (amino acids 57-421) was inserted into pET-28 (b) (between NcoI and XhoI) as a C-terminal His₆-tagged protein. Among six native cysteines of synaptotagmin I, five cysteines located in the transmembrane domain were changed to alanines. The triple mutant (K_326,327,331Q) was made using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The DNA sequences were confirmed by DNA sequencing (Iowa State University DNA Sequencing Facility, Ames, IA).

Protein Expression and Purification
The details of protein expression and purification were described previously (Lu et al., 2005; Xu et al., 2005). Briefly, GST-tagged proteins were expressed in Escherichia coli Rosetta (DE3) pLysS (Novagen, Madison, WI). GST fusion proteins were purified by affinity chromatography using glutathione-agarose beads (Sigma-Aldrich, St. Louis, MO). To purify proteins, the cell pellet was resuspended in 10 ml of PBST buffer [phosphate-buffered saline, pH 7.4, with 0.5% (vol/vol) Triton X-100] with the final concentrations of 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 1 mM EDTA, and 5 mM dithiothreitol (DTT). n-Lauroyl sarcosine (0.5%) was added to the solution. Cells were broken by sonication on the ice bath and centrifuged at 13,000 x g for 20 min at 4°C. The supernatant was mixed with 5 ml of glutathione-agrose beads (50%) in PBST buffer and incubated in the cold room for 2 h. During incubation for binding, 20 µg/ml DNase I and 4 µg/ml RNase were added. The beads were then washed with PBST buffer 10 times. The GST-tagged proteins were cleaved by bovine thrombin (Calbiochem, San Diego, CA) ng 1% n-octyl-D-glucopyranoside (OG).

The His-tagged synaptotagmin I was expressed in E. coli condon plus (BL21) pLysS (Stratagene) and induced by 0.3 mM isopropyl-D-galactopyranoside. The cell pellet was sonicated and centrifuged in 10 ml of lysis buffer (25 mM HEPES, 400 mM KCl, 10 mM imidazole, 2 mM EDTA, 2 mM AEBSF, 2 mM DTT, 0.5% Triton X-100, and 0.5% n-lauryl sarcosine, pH 7.4). The supernatant was mixed with 2 ml of nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (QIAGEN, Valencia, CA). DNase I and RNase were added to the supernatant and the Ni-NTA beads mixture. After binding for 2 h at 4°C, Ni-NTA beads were washed with wash buffer six times. Synaptotagmin I was eluted in elution buffer (25 mM HEPES, 400 mM KCl, 400 mM imidazole, and 1% OG, pH 8.0). Purified proteins were examined with 15% SDS–PAGE, and the purity was at least 90% for all proteins.

Membrane Reconstitution
The procedure was described previously (Lu et al., 2005, 2006). Briefly, syntaxin 1A was incubated with SNAP-25 for 1 h at room temperature to allow the formation of t-SNARE complex. The liposomes containing 1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) (molar ratio 85:15; 50 mM) were reconstituted with the preformed t-SNARE complex in a lipid/protein ratio of 200:1. The fluorescent liposomes containing POPC, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphoserine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-PS), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-PE) (molar ratio of 83:15:1:1; 10 mM) were reconstituted with VAMP2 in 200:1 lipid/protein ratio. Synaptotagmin I was reconstituted with VAMP2 (molar ratio 1:1). To remove OG, the samples were diluted two times with dialysis buffer (25 mM HEPES and 100 mM KCl, pH 7.4) and then dialyzed against 2 liters of dialysis buffer at 4°C overnight. After dialysis, the solution was centrifuged at 10,000 x g to remove protein and lipid aggregates. The reconstitution efficiencies were determined using SDS-PAGE and were at least 70%.

Total Fluorescence Lipid Mixing Assay
To measure the lipid mixing, v-SNARE liposomes with or without synaptotagmin I were mixed with t-SNARE liposomes in the ratio of 3:7. The final solution for each reaction contained 1 mM lipids in HEPES buffer (25 mM HEPES and 100 mM KCl, pH 7.4, and 5% glycerol) with the total volume of 100 µl. Fluorescence intensity was monitored with the excitation and emission wavelengths of 465 and 530 nm, respectively. The fluorescence signal was recorded using a Varian Cary Eclipse model fluorescence spectrophotometer with a quartz cell of 100 µl with a 2-mm path length. After 4000 s, 0.25% n-dodecylmaltoside was added to obtain the maximum fluorescence intensity. All of the lipid mixing experiments were carried out at 35°C.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synaptic Transmission Is Disrupted in Synaptotagmin Polylysine Motif Mutants
The C₂B polylysine motif of synaptotagmin I is required for efficient synaptic transmission (Figures 1, B and C, and 3; Mackler and Reist, 2001; Borden et al., 2005; Li et al., 2006). We examined excitatory junctional potentials (EJPs) in Drosophila synaptotagmin null third instars that expressed either a wild-type synaptotagmin transgene (–/–;P[syt^WT]) or a C₂B-polylysine motif mutant transgene (–/–;P[syt^KQ]). Figure 1B shows representative traces recorded in saline containing 1.5 mM Ca²⁺. Because both the polylysine motif transgene and the control transgene exhibit similar levels of synaptotagmin expression (Mackler and Reist, 2001), the only difference between these lines is the mutation in the C₂B polylysine motif of synaptotagmin. Figure 1C illustrates that the average EJP amplitude at third instar neuromuscular junctions in saline containing 1.5 mM Ca²⁺ was 14.2 ± 0.82 mV for polylysine motif mutants (–/–;P[syt^KQ]; n = 34 fibers) and 32.4 ± 0.99 mV for transgenic wild-type controls (–/–;P[syt^WT]; n = 42 fibers). Thus, similar to what has been reported previously for this synapse (Mackler and Reist, 2001), evoked transmitter release in 1.5 mM Ca²⁺ was decreased in polylysine motif mutants to 45% of transgenic controls.

The Postsynaptic Responsiveness Is Not Changed in Synaptotagmin Polylysine Motif Mutants
To verify that the decrease in the evoked response was not due to a developmental disruption that resulted in a reduced number of postsynaptic glutamate receptors or presynaptic active zones, we examined GluRIII (Marrus et al., 2004) and nc82 (an active zone marker; Wucherpfennig et al., 2003; Qin et al., 2005) staining in polylysine motif mutants and controls (Figure 2). The area of GluRIII staining [polylysine motif mutants, 49,500 ± 6600; transgenic controls, 47,000 ± 5500 pixels at 40x magnification (mean ± SEM)] was not significantly different in polylysine motif mutants compared with transgenic controls (p > 0.78, Student's t test; n = 6 junctions for each genotype). In addition, the amplitude of spontaneous fusion events is unchanged in the mutants (Mackler and Reist, 2001), demonstrating that the density of glutamate receptors is unchanged. Because neither the area nor density of postsynaptic receptors is changed, the postsynaptic response to neurotransmitter release is unaltered in the mutants. Furthermore, the area of nc82 staining [polylysine motif mutants: 65,200 ± 7500; transgenic controls: 63,800 ± 7600 pixels at 40x (mean ± SEM)] is also not significantly different in the mutants (p > 0.78, Student's t test; n = 6 junctions for each genotype). Thus, the decrease in the evoked response in polylysine motif mutants likely results from a defect in vesicle availability or release probability, because it does not result from a decrease in the ability of the postsynaptic cell to respond to neurotransmitter nor a decrease in the active zone area.

The Calcium Cooperativity of Release Is Unchanged in the Polylysine Motif Mutants, but the Apparent Calcium Affinity of Release Is Decreased
During synaptic transmission, the amount of neurotransmitter release triggered by nerve stimulation depends on some power (n) of the extracellular Ca²⁺ concentration. In 1967, Dodge and Rahamimoff showed at frog neuromuscular junctions that the dependence of release on extracellular Ca²⁺ can be explained by assuming a cooperative action of 4 Ca²⁺ ions (Dodge and Rahamimoff, 1967). Support for the hypothesis that the binding of three to five Ca²⁺ ions to a Ca²⁺ sensor(s) triggers neurotransmitter release has subsequently been demonstrated at other synapses as well, such as goldfish retinal bipolar synapses (Heidelberger et al., 1994), the rat Calyx of Held (Bollmann et al., 2000), and Drosophila neuromuscular junctions (Littleton et al., 1994; Stewart et al., 2000; Yoshihara and Littleton, 2002; Okamoto et al., 2005). To determine whether a disruption of Ca²⁺ binding by the polylysine motif mutant synaptotagmin contributed to the mutants' decreased release, we examined the Ca²⁺ dependence of release in both the polylysine motif mutants and the transgenic controls (Figure 3).

View larger version (14K): [in this window] [in a new window]   Figure 3. Calcium cooperativity of release is unchanged in the polylysine motif mutants, but the apparent calcium affinity and efficacy of release are decreased. EJPs were evoked by 0.05-Hz stimulation, and 10–15 sweeps were averaged for each fiber at each [Ca2+]. (A) EJP amplitudes at various extracellular Ca2+ concentrations for both polylysine motif mutants (gray triangles), and transgenic controls (black circles). For both genotypes, at 0.3–0.6 mM extracellular [Ca2+], n = 5–15 fibers; at 1.5 mM extracellular [Ca2+], n = 34–42 fibers; and at all other extracellular [Ca2+], n = 15–32 fibers. (B) Mean EJP amplitudes versus extracellular Ca2+ concentration were graphed on a double log plot. The slope (n) was determined by fitting the data points with linear regression lines (–/–;P[sytKQ]: n = 3.3, R = 0.99; –/–;P[sytWT]: n = 3.1, R = 0.95). (C) EJP amplitudes in A were normalized to the maximum value predicted by the Hill equation for each genotype and replotted to illustrate the shift in EC50 (–/–;P[sytKQ]: EC50 = 1.69 ± 0.08 mM; –/–;P[sytWT]: EC50 = 1.20 ± 0.05 mM). Error bars are SEM.

Although the Ca²⁺ cooperativity of release was not changed in the polylysine motif mutants, the EC₅₀ was changed. To illustrate the increase in EC₅₀ in the polylysine motif mutants, the EJP amplitudes were normalized to the predicted maximum value of the Hill equation for each genotype and replotted in Figure 3C [EC₅₀ = 1.69 ± 0.08 mM for –/–;P[syt^KQ] and EC₅₀ = 1.20 ± 0.05 for –/–;P[syt^WT] (mean ± SEM)]. Thus, the polylysine motif mutants exhibit an 40% increase in EC₅₀. Because the cooperativity of release is unchanged in these mutants, the increase in EC₅₀ corresponds to an 40% decrease in the apparent Ca²⁺ affinity of release. Similar results have been reported for cultured, hippocampal neurons expressing synaptotagmin with a mutation in the polylysine motif (Borden et al., 2005; Li et al., 2006).

Synaptotagmin Polylysine Motif Mutants Have a Decreased Release Probability
The finding that the mutants exhibit less release at every Ca²⁺ concentration indicates that the polylysine motif mutants have a decreased release probability and/or a decrease in the number of readily releasable vesicles. To examine release probability, we measured synaptic facilitation and augmentation during high frequency stimulation. Although the mechanisms underlying short-term plasticity are not completely understood, generally a decrease in release probability results in greater synaptic augmentation and facilitation (Atwood and Karunanithi, 2002; Zucker and Regehr, 2002). EJPs were evoked by 10-Hz stimulation for 30 s. Polylysine motif mutants exhibited larger synaptic augmentation than transgenic control larvae in 1.5 mM extracellular Ca²⁺ (Figure 4A) and still exhibited synaptic augmentation at 2.0 mM extracellular Ca²⁺, a concentration where control larvae no longer augment (Figure 4B). In addition, polylysine motif mutants exhibited facilitation during the first few pulses of 10 Hz stimulation in 1.5 mM Ca²⁺, but transgenic control larvae did not (Figure 4A, insets). Examination of the Ca²⁺ concentration where evoked release failed in the mutants also indicates a decrease in release probability. At 0.5 mM extracellular Ca²⁺, the failure rate in controls was 21 ± 5%, whereas that of the polylysine motif mutants was 64 ± 14% (p < 0.05). Thus, polylysine motif mutants exhibit a decreased release probability compared with transgenic control larvae.

View larger version (29K): [in this window] [in a new window]   Figure 4. Polylysine motif mutants have a decreased release probability compared with transgenic controls. EJPs were evoked by 30 s of 10-Hz stimulation in 1.5 mM (A) or 2.0 mM (B) extracellular Ca2+. EJP amplitudes were normalized to the amplitude of the EJP evoked by the first pulse and then averaged. After the first nine points, the points plotted are averages of 10 EJPs. Polylysine motif mutants: gray triangles, n = 6 fibers for 1.5 mM Ca2+ and n = 9 fibers for 2.0 mM Ca2+. Transgenic controls: black circles, n = 6 fibers for 1.5 mM Ca2+ and n = 10 fibers for 2.0 mM Ca2+. Error bars are SEM.

During a puff-application of 500 mM sucrose for 10 s, the frequency of miniature synaptic potentials (minis) dramatically increased in both polylysine motif mutants and transgenic controls (Figure 5). Figure 5A shows sample recordings from mutant and control larvae in both synaptic and nonsynaptic regions of the muscle fiber. The basal frequency of minis is elevated in each genotype only when sucrose is puffed on the synaptic region. The frequency before, during, and after the sucrose puff was determined and is plotted in Figure 5B. The basal frequency was higher in polylysine motif mutants than in controls (Figure 5B; Mackler and Reist, 2001). Therefore, to facilitate comparison between genotypes, the average frequency during the 10 s before the sucrose puff was subtracted from the calculated frequencies within each genotype (Figure 5C). The sucrose response in the polylysine motif mutants was comparable to that of controls. Thus, the decrease in the Ca²⁺-evoked response recorded in the polylysine motif mutants (Figure 3) is not due to a decrease in the size of the readily releasable pool of synaptic vesicles (Figure 5), but rather to a decrease in the release probability (Figure 4).

View larger version (20K): [in this window] [in a new window]   Figure 5. Size of the readily releasable pool of synaptic vesicles is unchanged in synaptotagmin polylysine motif mutants. (A) Sample traces showing that application of a hypertonic sucrose solution (bar) to synaptic regions for 10 s increases the frequency of miniature synaptic potentials. (B) Frequency of miniature synaptic potentials is plotted versus time from 10 s before to 10 s after the application of sucrose. (C) Because the basal rate of spontaneous release is greater in the mutants, the frequency of release was normalized to the basal rate within each genotype for comparison. The sucrose-induced, increased frequency in polylysine motif mutants (gray triangles, n = 9 fibers) is comparable to that in transgenic controls (black circles, n = 9 fibers). Error bars are SEM.

On Ca²⁺ influx, the interaction of synaptotagmin with the presynaptic membrane is thought to be important for fusion (Bai et al., 2000, 2002; Wang et al., 2003; Rhee et al., 2005). The Ca²⁺-independent interaction of the polylysine motif with t-SNARE heterodimers (Rickman et al., 2004) may play an important role in coupling synaptotagmin to the release machinery by positioning the Ca²⁺-binding site of the C₂B domain near the plasma membrane. This Ca²⁺-independent interaction would thus prime synaptic vesicles for efficient fusion. To directly test the importance of the C₂B polylysine motif in Ca²⁺-independent facilitation of fusion, we examined the rate of fusion in a fluorescence lipid mixing assay. When v-SNARE–containing liposomes are mixed with liposomes containing t-SNARE heterodimers in the absence of Ca²⁺, SNARE complex-specific fusion of the two populations occurred (Figure 6A; Weber et al., 1998; Parlati et al., 1999). Addition of membrane-bound synaptotagmin into the v-SNARE–containing liposomes directly accelerated the rate of fusion in a Ca²⁺-independent manner (Figure 6, A and B; Mahal et al., 2002) consistent with the hypothesis that a Ca²⁺-independent interaction between synaptotagmin and t-SNARE heterodimers facilitates SNARE-dependent fusion. Mutation of the polylysine motif, which has been shown to block the Ca²⁺-independent interaction between synaptotagmin and t-SNARE heterodimers (Rickman et al., 2004), abolished the ability of synaptotagmin to accelerate the fusion reaction (Figure 6, A and B). These results indicate that the polylysine motif of synaptotagmin plays a critical role in Ca²⁺-independent vesicle docking/priming and/or fusion in vitro.

View larger version (16K): [in this window] [in a new window]   Figure 6. Polylysine motif mutation abolishes synaptotagmin's Ca2+-independent ability to accelerate SNARE-mediated fusion. Fusion of v-SNARE liposomes (VAMP) with t-SNARE liposomes (syx/SNAP-25) was monitored via a fluorescence lipid mixing assay in the absence of Ca2+. (A) Sample traces showing the increase in fluorescence when t-SNARE liposomes are mixed with v-SNARE liposomes that do not contain synaptotagmin (syx/SNAP-25 + VAMP), with v-SNARE liposomes that contain wild-type synaptotagmin (syx/SNAP-25 + VAMP, sytWT) or with v-SNARE liposomes that contain the polylysine motif mutant synaptotagmin (syx/SNAP-25 + VAMP, sytKQ). Light gray trace shows the background rate of lipid mixing when SNAP-25 is omitted from the t-SNARE liposomes (syx + VAMP). Detergent is added at 4000 s to determine the maximum fluorescence intensity (Weber et al., 2000). (B) The final values before the addition of detergent were used. After subtraction of background fusion (syx + VAMP), values were normalized to fusion in the absence of synaptotagmin and plotted as fusion efficiency. n = 3; error bars are SD.

During 30 s of 10-Hz stimulation in 5 mM Ca²⁺, the larval neuromuscular junction exhibited synaptic depression (Figure 7). Both the sample traces in Figure 7A and the averaged data graphed in Figure 7B illustrate that during 30 s of stimulation, evoked release decreased to a greater extent in polylysine motif mutants than in transgenic controls. To compare the magnitude of depression in mutant and control transgenic lines, EJP amplitudes were normalized to the EJP amplitude elicited by the first pulse in the train. Despite that the polylysine motif mutants have a decreased release probability and release fewer vesicles with each stimulus (Figure 3, 5 mM Ca²⁺), they exhibited a small (6%) increase in the magnitude of depression (Figure 7B). However, this apparent increase in synaptic depression might be an artifact of nonlinear summation. Because the EJP amplitudes are larger in the controls (Figures 3A and 7A), they would be more affected by nonlinear summation, which would underestimate their true degree of synaptic depression relative to the mutants. To explore this possibility, we applied the equation of Martin (1955) as an estimate to correct for nonlinear summation (our unpublished data). Indeed, using this correction factor, the polylysine motif mutants exhibited slightly less (4%) synaptic depression than controls. However, because this formula is an imperfect correction (McLachlan and Martin, 1981), the true degree of synaptic depression is likely somewhere between that calculated from the noncorrected and corrected EJP amplitudes. Thus, the degree of synaptic depression is likely quite similar between mutants and controls.

View larger version (32K): [in this window] [in a new window]   Figure 7. Polylysine motif mutants recover more slowly from synaptic depression than transgenic controls. Synaptic depression was elicited by stimulating fibers at 10 Hz for 30 s in 5 mM extracellular Ca2+. Recovery was monitored by recording the EJP amplitude of a single shock either 5 or 15 s after the 10-Hz stimulation stopped. Each fiber was also stimulated at an extended time point (140 s) to verify full recovery. (A) Representative traces from polylysine motif mutants (–/–;P[sytKQ]) and transgenic controls (–/–;P[sytWT]) showing the first and last 3.5 s of stimulation at 10 Hz for 30 s in saline containing 5 mM Ca2+. (B) EJP amplitudes were normalized to the amplitude of the first shock. After the first nine points, the points plotted during the 10-Hz stimulation are averages of 10 EJPs. Transgenic controls: black circles, n = 10 fibers for 5-s recovery and n = 14 fibers for 15-s recovery. Polylysine motif mutants: gray triangles, n = 10 fibers for 5-s recovery and n = 14 fibers for 15-s recovery. Error bars are SEM.

Synaptic Vesicle Endocytosis Is Not Impaired in Polylysine Motif Mutants
Our in vitro studies demonstrate a Ca²⁺-independent role for the polylysine motif in docking/priming and/or fusion, whereas our in vivo studies demonstrate a Ca²⁺-independent role before Ca²⁺-triggered fusion. Together, these studies support a role for the polylysine motif in docking/priming; however, they do not exclude additional disruptions during synaptic vesicle recycling. Recycling consists of many steps that include, but are not limited to, endocytosis, clathrin uncoating, refilling with neurotransmitter, docking, and priming of vesicles. Because functional studies demonstrate that synaptotagmin I is required during endocytosis (von Poser et al., 2000; Jarousse and Kelly, 2001; Jarousse et al., 2003; Poskanzer et al., 2003; Llinás et al., 2004; Nicholson-Tomishima and Ryan, 2004) and the polylysine motif of the C₂B domain of synaptotagmin is postulated to mediate this role (Takei and Haucke, 2001; but see Poskanzer et al., 2006), we wanted to determine whether endocytosis was disrupted in our mutants. However, the rate of endocytosis is difficult to assess in mutants with defects in exocytosis. Therefore, we investigated the importance of the C₂B polylysine motif for endocytosis by using three approaches.

As a first step in examining endocytosis, the ability of the mutants to maintain a supply of synaptic vesicles for release was further challenged by stimulating preparations at 10 Hz for 4 min in 5 mM Ca²⁺. The averaged data (Figure 8A) illustrate that during the 4 min of stimulation evoked release decreased to a greater extent in polylysine motif mutants than in transgenic controls. To compare the magnitude of depression in mutant and control transgenic lines, EJP amplitudes were binned at 5-s intervals and normalized to the first EJP amplitude bin (Figure 8B). EJP amplitudes from polylysine motif mutants did decrease to a greater extent than the transgenic controls. Although this result seems to suggest that the rate of synaptic vesicle resupply relative to the rate of exocytosis is slowed in the polylysine motif mutants compared with controls, again this apparent difference is probably exaggerated by nonlinear summation. Indeed, when the correction factor of Martin (Martin, 1955) is applied, the polylysine motif mutants again show less depression (4%) than controls (our unpublished data). Even without the correction factor, the increased synaptic depression in the polylysine motif mutants is not severe (5% more depression after 60 s and 15% more after 4 min). Thus, the polylysine motif mutants are able to maintain release relatively well for at least 4 min of 10-Hz stimulation, an unexpected result if the polylysine motif were critical for endocytosis.

View larger version (17K): [in this window] [in a new window]   Figure 8. Polylysine motif mutants maintain significant levels of evoked release during prolonged high-frequency stimulation. (A) Average EJP amplitudes evoked during 4 min of 10-Hz stimulation in saline containing 5 mM Ca2+. Except for the first nine points and the last point, the EJP amplitudes were binned at 5-s intervals, and the average of the bin is plotted. The first nine points are not binned. The last point is the average EJP amplitude of a 3-s bin. Transgenic controls: black circles, n = 8 fibers. Polylysine motif mutants: gray triangles, n = 10 fibers. (B) Same EJP amplitudes as in A but binned at 5-s intervals and normalized to the first 5-s bin.

View larger version (123K): [in this window] [in a new window]   Figure 9. Synaptic vesicles are abundant in nerve terminals of polylysine motif mutants. Electron micrographs showing the synaptic ultrastructure of neuromuscular junctions in transgenic controls (top), polylysine motif mutants (middle), and sytnull mutants (bottom). Bar, 200 nm.

Finally, to examine the rate of endocytosis in polylysine motif mutants directly, we used an FM 1-43 dye uptake assay. FM 1-43 fluoresces when it inserts into lipid membranes. If it is present when synaptic vesicle membrane is internalized, these synaptic vesicles will be fluorescently labeled. Subsequently, the dye can be washed from the outside of cells, leaving only the internalized synaptic vesicles labeled. Thus, FM 1-43 makes it possible to examine the process of endocytosis directly (Betz et al., 1992).

Figure 10A illustrates the endocytic assay we used to test the involvement of the polylysine motif in endocytosis [adapted from the assays of (Ryan et al., 1996; Kuromi and Kidokoro, 1998; Stimson et al., 2001; Kim et al., 2002)]. Drosophila third instars were stimulated for 6 min with a stimulating saline containing high potassium. Stimulation was terminated when the stimulating saline was replaced by standard saline plus 3 µM TTX. TTX was included to block any spontaneous action potentials coming from the attached CNS. The standard saline + TTX was left on the preparation for a variable amount of time (t = 0, 30, 60, or 180 s). After t, the preparation was bathed in standard saline containing TTX and FM 1-43 for 6 min to label endocytosing vesicles. Finally the preparation was washed for 15 min with 3 changes of Ca²⁺-free saline to remove extracellular FM 1-43 from the plasma membrane. Ca²⁺-free saline was used to minimize vesicle fusion during the washing stage. After washing, the fluorescence intensity of the FM 1-43-labeled synaptic boutons was measured.

View larger version (34K): [in this window] [in a new window]   Figure 10. Rate of endocytosis is not significantly different in synaptotagmin polylysine motif mutants. (A) Schematic of endocytic assay using FM 1-43 dye. (B) Synaptic boutons on muscle 6 and 7 in polylysine motif mutants (–/–;P[sytKQ]) and transgenic controls (–/–;P[sytWT]) were labeled with FM 1-43 dye as described in A. Labeling is shown for t = 0 s (top) and t = 180 (bottom). (C) At t = 0, the average fluorescence intensity of boutons from polylysine motif mutants was 10% less than controls. (D) Fluorescence intensity of synaptic boutons is graphed as a function of the time between stimulus termination and application of the FM 1-43 dye (t). Polylysine motif mutants: gray triangles, n = 132–135 boutons from nine larvae for each t. Transgenic controls: black circles, n = 135 boutons from nine larvae for each t. To account for differences between genotypes in the absolute number of synaptic vesicles released during stimulation, fluorescence values for each genotype were normalized to the fluorescence intensity of that genotype at t = 0 s. 95% CI = –11.6 to 2.75.

During the delay period between the end of stimulation and addition of FM 1-43 dye, t, some fraction of the synaptic vesicle membrane remaining on the plasma membrane undergoes endocytosis. As t increases, more and more vesicle membrane is internalized, leaving less vesicle membrane on the surface to be labeled when FM 1-43 is subsequently applied. Therefore, as t increases, less FM 1-43 is internalized and nerve terminal labeling becomes progressively dimmer. This is illustrated in Figure 10B, which shows FM 1-43-labeled synaptic boutons on muscle 6 and 7 in both transgenic control and polylysine motif mutant larvae after t = 0 and 180 s. In both polylysine motif mutant and transgenic control larvae, labeling was brightest at t = 0 s and dimmest at t = 180 s. Loading was dependent on both stimulation and Ca²⁺ (our unpublished data).

At all time points, the mean fluorescence of polylysine motif mutant boutons was somewhat lower than the mean fluorescence for controls. This is most likely because the mutants exocytose fewer vesicles than the controls during the stimulation. Figure 10C shows the mean level of FM 1-43 labeling at t = 0 s for both mutants and controls. Under these stimulation conditions (high K⁺, high Ca²⁺ for 6 min), the amount of membrane awaiting endocytosis after stimulation ceased was only 10% less in the polylysine motif mutants. Because exocytosis is 40% less in the mutants in 5 mM Ca²⁺ (Figure 3A), this relative buildup of vesicle membrane stranded in the plasma membrane could indicate that endocytosis is slowed in the mutants. However, a single time point cannot accurately measure the rate of endocytosis because other factors, such as the size of the releasable pools and saturation of the endocytic machinery, can also influence the amount of membrane awaiting endocytosis. So, to determine the kinetics of synaptic vesicle internalization independent of this 10% difference, we normalized our data according to previous methods used to compare endocytic rates between preparations with different amounts of exocytosis (Ryan et al., 1996; Stimson et al., 2001; Kim et al., 2002). Thus, the fluorescence value of each bouton was normalized to the mean fluorescence value of boutons of the corresponding genotype at t = 0 s.

If the rate of endocytosis were impaired in polylysine motif mutant larvae, then during any given t, they should not be able to endocytose as large a fraction of the remaining synaptic vesicle membrane as controls. As a result, a larger proportion of synaptic vesicle membrane would remain in the plasma membrane when the FM 1-43 was applied, leading to proportionally more FM 1-43 internalization. Thus, if polylysine motif mutants had endocytic deficits, then after any given t, polylysine motif terminals should exhibit a normalized fluorescence that was brighter than controls. Indeed, stoned mutants, which also exhibit a decrease in evoked release, were shown to have an endocytic defect using a similar assay (Stimson et al., 2001). C₂B polylysine motif mutants, in contrast, do not (Figure 10D). At each time point, the normalized fluorescence of the mutant terminals was slightly lower than that of controls. Lower fluorescence values would suggest a faster rate of endocytosis in the mutants, not an impaired rate. However, the differences between polylysine motif mutants and transgenic controls were not statistically significant. The mixed model ANOVA used to generate a 95% confidence interval for the true difference between mutants and controls included 0 (–11.6, 2.75). Because the rate of endocytosis was not disrupted in the mutants, the relatively small difference in labeling at t = 0 s (Figure 10C) suggests that endocytosis was the rate-limiting step and that our strong stimulation protocol successfully drove the majority of the cycling vesicle membrane (which is approximately equal in mutants and controls; Figure 5C) to the surface.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The highly conserved C₂B polylysine motif plays a functional role in the synaptic vesicle cycle. Mutations in this motif decrease the amplitude of the evoked response at all Ca²⁺ concentrations; increase the EC₅₀ of Ca²⁺ for release; increase augmentation, facilitation, and failure rate at low Ca²⁺ concentrations; cause a marked decrease in the rate of recovery from synaptic depression; block Ca²⁺-independent interactions with t-SNARE heterodimers (Rickman et al., 2004); and abolish the ability of synaptotagmin to accelerate Ca²⁺-independent, SNARE-mediated fusion. However, polylysine motif mutations do not alter postsynaptic receptor area, the size of minis (Mackler and Reist, 2001), active zone area, the Ca²⁺ cooperativity of release, the size of the hypertonic response, or the rate of endocytosis, and they do not result in synaptic vesicle depletion. These findings are discussed in terms of a single deficit hypothesis, namely, disruption of Ca²⁺-independent synaptic vesicle docking/priming (Figure 11).

View larger version (19K): [in this window] [in a new window]   Figure 11. Model of the role of the polylysine motif in Ca2+-independent synaptic vesicle docking/priming. Nuclear magnetic resonance structures of C2A (PDB file 1BYN) and C2B (PDB file 1K5W) domains (yellow) of synaptotagmin, the crystal structure of the core complex (PDB file 1SFC, containing VAMP [blue], SNAP-25 [green], and syntaxin [red]), and Ca2+ (pink) are shown to scale by using the PyMOL Molecular Graphics System (DeLano, 2002). The N termini of the SNARE proteins are darker and the C termini are paler to indicate depth. The membranes, the link between C2A and C2B domains of synaptotagmin, as well as transmembrane links for synaptotagmin, VAMP, syntaxin, and SNAP-25 were subsequently added. (A) Ca2+-independent interactions between the polylysine motif lysines in C2B (yellow, space-filled residues) with negatively charged residues of SNAP-25 (green, space-filled residues; Rickman et al., 2004, 2006) could hold the C2B Ca2+ binding site in the immediate vicinity of negatively charged phospholipi