Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Mar 30;367(3):848-63.
doi: 10.1016/j.jmb.2007.01.040. Epub 2007 Jan 23.

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

Han Dai  1 , Nan Shen, Demet Araç, Josep Rizo
Affiliations

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

Han Dai et al. J Mol Biol. .

Abstract

The function of synaptotagmin as a Ca(2+) sensor in neurotransmitter release involves Ca(2+)-dependent phospholipid binding to its two C(2) domains, but this activity alone does not explain why Ca(2+) binding to the C(2)B domain is more critical for release than Ca(2+) binding to the C(2)A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca(2+) induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca(2+) binding to the C(2)B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C(2)B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The C2AB fragment binds preferentially to membranes containing SNARE complexes. (a) Diagram summarizing the microfluidic channel experiments designed to test whether the C2AB fragment partitions preferentially to membranes containing reconstituted SNARE complexes formed by the syntaxin SNARE motif and TM region (yellow), the synaptobrevin SNARE motif (red) and the SNAP-25 N-terminal (blue) and C-terminal (green) SNARE motifs (molar protein to lipid ratio 1:1000). The V419C-C2AB fragment is represented by two orange elipses with a green star depicting the site labeled with BODIPY-FL. The same color coding is used in all figures. See text for further details. (b) Confocal micrographs of supported bilayers (41% POPC, 32% DPPE, 12% DOPS, 5% PI and 10% Cholesterol [w/w]) containing or lacking reconstituted SNARE complexes, which where deposited in the external microchannels of the PDMS slab (0.2 mm width). The micrographs were obtained after injecting 0.5 equivalents of BODIPY-FL labeled V419C-C2AB fragment (40 nM concentration) in buffer containing 1 mM EDTA through the central microchannel with the valved screw open, incubating for 1 hr and washing the microchannels with the same buffer. (c) Analogous experiments performed with buffer containing 1 mM Ca2+ and 0.5 equivalents of BODIPY-FL labeled V419C-C2AB fragment. (d) Quantitative analysis of the data shown in (b,c). Average fluorescence intensities were measured in each microchannel. The average intensity observed for the residual binding to SNARE-free membranes in the absence of Ca2+ (right panel in (b)) was then subtracted and used to normalize all resulting values. Error bars reflect standard deviations. (e) Partition experiment performed as in (c) but with 2 equivalents of BODIPY-FL labeled V419C-C2AB fragment.
Figure 2
Figure 2
The Ca2+-binding loops of the synaptotagmin 1 C2 domains remain inserted into the membrane in the presence of reconstituted SNARE complexes. (a) Fluorescence spectra of NBD-F234C mutant C2AB fragment in the presence of phospholipid vesicles lacking (black traces) or containing (red traces) reconstituted SNARE complexes, acquired in 1 mM EDTA or 1 mM Ca2+. The bottom panel shows analogous spectra acquired in the presence soluble SNARE complexes and 1 mM EDTA (green) or 1 mM Ca2+ (magenta). (b) Analogous experiments performed with NBD-V304C mutant C2AB fragment. The presence of phospholipid vesicles does not perturb the fluorescence spectra of both isolated C2AB fragments in 1 mM EDTA.
Figure 3
Figure 3
NMR analysis of Ca2+-independent interactions between synaptotagmin 1 and the SNARE complex. (a) Superposition of 1H-15N HSQC spectra of Ca2+-free 2H,15N-labeled C2AB fragment in the absence (black contours) and presence (red contours) of unlabeled short SNARE complex. (b) Expansions of the 1H-15N HSQC spectra shown in (a). Cross-peaks from the C2B domain are labeled with the residue number and one letter abbreviation, whereas cross-peaks from the C2A domain are labeled with a blue ‘A’. (c) Ribbon diagram of the C2B domain summarizing the most significant perturbations in the 1H-15N HSQC cross-peaks of the C2B domain caused by Ca2+-independent binding to the short SNARE complex. Residues corresponding to cross-peaks that exhibit significant shifts or have strong initial intensities and are broadened beyond detection upon binding are colored in cyan (disappearance of cross-peaks with weak initial intensities was not considered significant due to the overall broadening observed for most C2B domain cross-peaks). The two Ca2+ ions that bind to the C2B domain are shown as blue spheres to point out their binding sites, but the data were obtained in the absence of Ca2+. Strand 4 of the β-sandwich is labeled. (d) Superposition of 1H-15N TROSY-HSQC spectra of a sample of the short SNARE complex with the C-terminal SNAP-25 SNARE motif 2H,15N-labeled, acquired in 1 mM EDTA and the absence (black contours) or presence (red contours) of unlabeled C2AB fragment. Cross-peaks exhibiting the most significant broadening upon binding are labeled. The cross-peak assignments were described previously. (e) Ribbon diagram of the SNARE complex with the SNAP-25 residues corresponding to severely broadened cross-peaks shown in white.
Figure 4
Figure 4
Structural determinants of SSCAP complex formation. (a) Confocal micrographs of supported bilayers containing reconstituted SNARE complexes deposited within microfluidic channels (width: 0.2 mm). The bilayers were loaded with 50 nM BODIPY-FL-labeled complexin fragment (residues 26–83) and washed with standard buffer containing 1 μM C2AB fragment (+Syt) and 1 mM EDTA (-Ca2+) or 1 mM Ca2+ (+Ca2+). The membranes contained 41% POPC, 32% DPPE, 12% DOPS, 5% PI and 10% Cholesterol [w/w] (two left images) or 58% POPC, 32% DPPE, and 10% Cholesterol [w/w] (right image). (b),(c) Displacement of BODIPY-FL-labeled complexin fragment from membrane anchored wild-type and mutant SNARE complexes upon addition of different concentrations of wild-type and mutant C2AB fragments. Experiments were performed as in (a) in 1 mM Ca2+ with membranes containining 41% POPC, 32% DPPE, 12% DOPS, 5% PI and 10% Cholesterol [w/w]. Average fluorescence intensities measured under each condition were normalized to a control in which the fluorescently labeled complexin fragment was added to a supported bilayer lacking SNARE complexes. Error bars represent SEMs derived from three separate measurements. In (b) all experiments were performed with wild type SNARE complexes and additions of wild type C2AB fragment (magenta), C2A domain (black) or C2B domain (red), or mutant C2AB fragments bearing mutations that disrupt Ca2+- binding to the C2A domain (D178N, green) or the C2B domain (D309N, blue). In (c), experiments were performed with wild type SNARE complexes and C2AB fragment bearing the K326A,K327A mutation in the polybasic region of the C2B domain (green), or with wild type C2AB fragment and SNARE complexes bearing mutations in acidic residues of SNAP-25 (D51K,E52K,E55K blue; D186K,D193K, light green); the data obtained with wild type SNARE complexes and the C2AB fragment (magenta), C2A domain (black) or C2B domain (red) are also shown in this diagram for comparative purposes.
Figure 5
Figure 5
Model of the SSCAP complex. (a) Ribbon diagrams of the five top HADDOCK models. The five models were superimposed using the coordinates of the SNARE complex to illustrate the variability in the relative orientation of the C2B domain. (b) Ribbon diagram of the HADDOCK model that is most similar to the model obtained with FTDOCK, in an orientation rotated 90° with respect to that of (a). The two Ca2+ ions that bind to the C2B domain, which were not included in the modeling calculations, are represented as blue spheres, and the membrane phospholipids are indicated in light gray to help visualizing the relative locations of all the components of the SSCAP complex. N and C denote the N- and C-termini of the C2B domain and the SNARE complex. The residues of the SNAP-25 SNARE motif corresponding to cross-peaks that were perturbed by C2AB fragment binding in our NMR experiments (Figure 3) are colored in white. (c) Ribbon diagram of the same HADDOCK model shown in B but in a different orientation and including the C2A domain to illustrate how it emerges from the N-terminus of the C2B domain away from the SNARE complex. The linker sequence between the two C2 domains (four residues) is represented by a dashed orange curve. Residues that were mutated in our complexin displacement assays of Figure 4C are shown as spheres: K326 and K327 of the C2B domain are in blue; D51, E52, E55, D186 and D193 of SNAP-25 are in pink. D51, E522 and E55 are also shown in (B). (d) Superposition of the crystal structure of complexin bound to the SNARE complex with the model of the C2B domain/SNARE complex shown in (b). Partially transparent surfaces of complexin and the C2B domain are shown to illustrate the limited overlap between their binding sites on the SNARE complex. The color-coding for the SNAREs is the same as in Figure 1(a), synaptotagmin is in orange and complexin in pink. The models were rendered with PyMol (DeLano Scientific, San Carlos, CA).
Figure 6
Figure 6
Models illustrating how the SSCAP complex could trigger neurotransmitter release. (a,b) The diagrams show the surface electrostatic potential of two copies of the modeled C2B domain/SNARE complex and how the highly positive potential at the tips of the complexes could help to bend the membranes to initiate membrane fusion (a) or could help opening the fusion pore (b). The model on the left was slightly rotated to better show the highly positive surface at the tip. The model on the right is rotated 180° around the vertical axis with respect to that on the left. The TM regions of syntaxin and synaptobrevin are in yellow and red, respectively. The electrostatic surface potential was computed with GRASP. The models were rendered with PyMol (DeLano Scientific, San Carlos, CA). The electrostatic potential is contoured at the 5 kT/e level, with red denoting negative potential and blue denoting positive potential.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sudhof TC. The synaptic vesicle cycle. Annu Rev Neurosci. 2004;27:509–547. - PubMed
    1. Fernandez-Chacon R, Konigstorfer A, Gerber SH, Garcia J, Matos MF, Stevens CF, Brose N, Rizo J, Rosenmund C, Sudhof TC. Synaptotagmin I functions as a calcium regulator of release probability. Nature. 2001;410:41–49. - PubMed
    1. Mackler JM, Drummond JA, Loewen CA, Robinson IM, Reist NE. The C(2)B Ca(2+)-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature. 2002;418:340–344. - PubMed
    1. Rhee JS, Li LY, Shin OH, Rah JC, Rizo J, Sudhof TC, Rosenmund C. Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1. Proc Natl Acad Sci USA. 2005;102:18664–18669. - PMC - PubMed
    1. Bai J, Chapman ER. The C2 domains of synaptotagmin--partners in exocytosis. Trends Biochem Sci. 2004;29:143–151. - PubMed

Publication types

MeSH terms

LinkOut - more resources