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. 2010 May;17(5):568-75.
doi: 10.1038/nsmb.1791. Epub 2010 Apr 18.

Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity

Mingshan Xue  1 , Timothy K Craig, Junjie Xu, Hsiao-Tuan Chao, Josep Rizo, Christian Rosenmund
Affiliations

Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity

Mingshan Xue et al. Nat Struct Mol Biol. 2010 May.

Abstract

Complexins facilitate and inhibit neurotransmitter release through distinct domains, and their function was proposed to be coupled to the Ca(2+) sensor synaptotagmin-1 (Syt1). However, the mechanisms underlying complexin function remain unclear. We now uncover an interaction between the complexin N terminus and the SNARE complex C terminus, and we show that disrupting this interaction abolishes the facilitatory function of complexins in mouse neurons. Analyses of hypertonically induced exocytosis show that complexins enhance synaptic-vesicle fusogenicity. Genetic experiments crossing complexin- and Syt1-null mice indicate a functional interaction between these proteins but also show that complexins can promote Ca(2+)-triggered release in the absence of Syt1. We propose that the complexin N terminus stabilizes the SNARE complex C terminus and/or helps release the inhibitory function of complexins, thereby activating the fusion machinery in a manner that may cooperate with Syt1 but does not require Syt1.

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Figures

Figure 1
Figure 1
The N terminus of CplxI facilitates Ca2+-triggered neurotransmitter release. (a) Schematic diagram of CplxI domains. Numbers above the diagram indicate the boundary residues. (b,c,f) Representative traces of Cplx-KO neurons and Cplx-KO neurons rescued by CplxI WT, CplxI 16–134, or CplxI 8–134. The arrows represent stimulations. Artifacts and action potentials are blanked. (b) Basal evoked EPSCs. (c) Five consecutive EPSCs evoked at 20 ms inter-stimulus interval (ISI). (f) Evoked EPSCs in standard external solutions (4 mM Ca2+, 4 mM Mg2+) and in solutions with high Ca2+ concentrations (12 mM Ca2+, 1 mM Mg2+) or low Ca2+ concentrations (1 mM Ca2+, 1 mM Mg2+). (d,e,g,h) Bar graphs show the summary data of paired-pulse ratio (d), Pvr (e), EPSC amplitude potentiation by elevating Ca2+ concentration (g), and EPSC amplitude depression by lowering Ca2+ concentration (h). Data are normalized to the mean values of the corresponding CplxI WT rescue (black dashed lines and error bars). Data are expressed as mean ± SEM. **, P < 0.001; ***, P < 0.0001 compared to the corresponding CplxI WT rescue. The numbers of neurons analyzed are indicated on the bars.
Figure 2
Figure 2
Identification of Met5 and Lys6 as crucial residues for CplxI N-terminal function. (a) Helical wheel model of rat CplxI N terminus. Residues are numbered and labeled as orange (hydrophobic), red (negatively charged), and blue (positively charged). (b) N terminus amino acid sequence alignment of Complexin paralogs and orthologs. Identical residues are marked as red and highly conserved residues are marked as blue. Hs, Homo sapiens; Rn, Rattus norvegicus; Mm, Mus musculus; Lp, Loligo pealeii; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans. Asterisks mark residues Met5 and Lys6 in RnCplxI. (c–f) Summary data of evoked release from Cplx-KO neurons and Cplx-KO neurons rescued by WT or mutant CplxI. Bar graphs show paired-pulse ratio (c), Pvr (d), EPSC amplitude potentiation by elevating Ca2+ concentration (e), and EPSC amplitude depression by lowering Ca2+ concentration (f). Data are normalized to the mean values of the corresponding CplxI WT rescue (black dashed lines and error bars). Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0001 compared to the corresponding CplxI WT rescue. The numbers of neurons analyzed are indicated on the bars.
Figure 3
Figure 3
The CplxI N terminus facilitates spontaneous release. (a) Representative traces of mEPSCs from Cplx-KO neurons and Cplx-KO neurons rescued by WT or mutant CplxI. (b,c) Bar graphs show mEPSC frequency (b) and amplitude (c). Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to CplxI WT-rescued Cplx-KO neurons. The numbers of neurons analyzed are indicated on the bars.
Figure 4
Figure 4
The CplxI N terminus binds to the SNARE complex and the M5E K6E mutation disrupts this interaction. (a,d,e) Expansions of 1H-15N HSQC spectra of WT CplxI alone (a,d,e, black contours) or in the presence of SNARE complex-containing proteoliposomes (a, red contours), plain liposomes (d, green contours), or soluble SNARE complex (e, red contours). Selected cross-peaks are labeled. Cross-peaks from residues arising from the expression vector are indicated with asterisks. (b) Expansions of 1H-15N HSQC spectra of WT CplxI alone (black contours) and CplxI M5E K6E alone (blue contours). New peaks from CplxI M5E K6E are labeled with “N”. (c,f) Expansions of 1H-15N HSQC spectra of CplxI M5E K6E alone (c,f, blue contours) or in the presence of SNARE complex-containing proteoliposomes (c, orange contours) or soluble SNARE complex (f, orange contours). (g–j) Bar diagrams showing the relative cross-peak intensities of 1H-15N HSQC spectra of CplxI alone (g) and after adding SNARE complex-containing proteoliposomes (h), plain liposomes (i), or soluble SNARE complex (j). The corresponding spectra are shown in panels (a,d,e). The relative intensities for each spectrum are obtained by dividing absolute intensities by the average of all cross-peak intensities. Only well-resolved cross-peaks are quantified. Cross-peaks that disappeared below the noise level are assigned zero intensity. Note that, because of limited sensitivity under the conditions of our experiments, the individual cross-peak intensities exhibit substantial variability in repeated experiments, but the overall relative intensity patterns conclusively illustrate the conclusions drawn in the text.
Figure 5
Figure 5
The CplxI N terminus binds to the C terminus of the SNARE complex. (a) Expansions of 1H-15N TROSY-HSQC spectra of SNARE complex with the SNAP-25 C-terminal SNARE motif 2H,15N-labeled, in the presence of CplxI 2–82 A12C-MTSL before (red contours) and after (black contours) reduction with dithionite. Selected cross-peaks are labeled with the residue number. (b) Ribbon diagram of the crystal structure of the CplxI 26–83/SNARE complex illustrating the PBEs caused by CplxI 2–82 A12C-MTSL on the 1H-15N TROSY-HSQC cross-peaks of the SNARE complex. CplxI is colored pink (central α-helix) and orange (accessory α-helix), Synaptobrevin-2 red, Syntaxin-1 yellow, and SNAP-25 blue and green. Residues in black correspond to well-resolved cross-peaks whose intensities decrease by more than 50% due to the PBEs induced by MTSL (residues 78, 81, 82, 86, 87 and 90 of Synaptobrevin-2, residues 242 and 247 of Syntaxin-1, and residues 73-75, 78, 80, 195, 196, 198–204 of SNAP-25). All these residues are clustered at the C terminus of the SNARE complex, and the cross-peaks from other residues in this region are not observable or overlapped. We estimate that the amide protons of these residues are approximately within 18 Å or less from the probe. The asterisk indicates the estimated position of the MTSL probe in the bound CplxI 2–82 A12C-MTSL.
Figure 6
Figure 6
Complexins regulate synaptic vesicle fusogenicity. (a) Average traces of synaptic responses induced by sucrose solutions from control (500 mM, n = 82; 250 mM, n = 27), CplxI/II-DKO (500 mM, n = 78; 250 mM, n = 25), and CplxI/II/III-TKO (500 mM, n = 78; 250 mM, n = 28) neurons. (b,c) Summary data of 250 mM sucrose solution-induced response onset latency (b), peak release rate and fraction of RRP released (c) from control, CplxI/II-DKO, and CplxI/II/III-TKO neurons. Data are expressed as mean ± SEM; *, P < 0.05; ***, P < 0.001 compared to control. Control neurons are either CplxII−/− or CplxII−/− CplxIII+/−. (d,e) Summary data of 250 mM sucrose solution-induced response onset latency (d), peak release rate and fraction of RRP released (e) from Cplx-KO neurons and Cplx-KO neurons rescued by WT or mutant CplxI variants. Data are expressed as mean ± SEM; **, P < 0.01; ***, P < 0.001 compared to CplxI WT-rescued Cplx-KO neurons. The numbers of neurons analyzed are indicated in the figures.
Figure 7
Figure 7
Complexins facilitate Ca2+-evoked neurotransmitter release independently and cooperatively with Synaptotagmin-1. (a) Representative evoked EPSC traces of neurons from various genotypes. The arrows represent stimulations. Artifacts and action potentials are blanked. (b,c) Bar graphs show EPSC amplitude (b) and Pvr of evoked release (c). CplxII-KO neurons serve as control, as they are indistinguishable from WT neurons. Data are normalized to the mean values of control neurons. Genotypes are indicated below the bars. (d) Pvr of evoked release from WT and Syt1 heterozygous neurons. (e) Pvr of evoked release from CplxI/II-DKO and Syt1-Het/CplxI-KO/CplxII-KO neurons. Data are expressed as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to control neurons (b,c) or to CplxI/II-DKO neurons (e). The numbers of neurons analyzed are indicated above the bars.
Figure 8
Figure 8
Proposed model for the key facilitatory function of the Complexin N terminus. (a) The SNARE complex (Synaptobrevin-2, red; Syntaxin-1, yellow; and SNAP-25, green) assembles partially during priming. “N” and “C” indicate the N and C termini, respectively. (b–d) Complexins bind to the SNARE complex in at least two different modes that correspond to an inhibited (b) and an activated state (c,d). (b) The Complexin central α-helix binds to the partially assembled SNARE complex and helps to assemble the SNARE complex further. At the same time, the Complexin accessory α-helix replaces the C terminus of the Synaptobrevin-2 SNARE motif in the four-helix bundle, preventing C-terminal assembly,. (c) The Complexin accessory α-helix is released from the SNARE complex by the Complexin N terminus, Synaptotagmin-1/Ca2+, or both, allowing full assembly of the SNARE complex C terminus. (d) The SNARE complex is fully assembled. Binding of the Complexin N terminus to the SNARE complex C terminus is proposed to help releasing the inhibition of the accessory α-helix and/or to stabilize the C terminus of the SNARE complex to assist in exerting force on the membranes. Note that the location of the Complexin N terminus is only tentative in (c) and (d). The Complexin C terminus is not shown for simplicity.
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