Selective Interaction of Complexin with the Neuronal SNARE Complex

Pabst, Stefan; Hazzard, James W.; Antonin, Wolfram; Südhof, Thomas C.; Jahn, Reinhard; Rizo, Josep; Fasshauer, Dirk

doi:10.1074/jbc.m002571200

Cited by 169 publications

(211 citation statements)

References 43 publications

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“…These results are partially supported by evidence that syntaxin 2 coimmunoprecipitated with VAMP 2 from acinar cell membranes (36). Moreover, when evaluated in vitro using recombinant proteins, no association of complexin 1 or 2 with SNARE complexes containing syntaxin 4 was found (13,21), suggesting that the interaction with syntaxin 4 seen in acini may involve additional regulatory proteins.…”

Section: Discussionsupporting

confidence: 69%

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Falkowski

Thomas

Groblewski

2010

Journal of Biological Chemistry

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Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca 2؉ -stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central ␣-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central ␣-helical domain did not alter SNARE binding and moreover, augmented Ca 2؉ -stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca 2؉ -sensitive regulatory pathway for zymogen granule exocytosis.Complexin 1, originally identified as synaphin, is a small cytosolic protein that associates with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, which are essential to drive membrane fusion during neurotransmitter exocytosis (1, 2). Although complexin 1 is expressed exclusively in neurons, complexin 2 appears to be ubiquitously expressed and presumably functions in other secretory cell systems. Complexins 1 and 2 are highly homologous, each containing 134 amino acids that are 86% identical (3). Moreover, rat, mouse, and human complexin 2 proteins are 100% identical, although the nucleotide sequences of their mRNAs are considerably different (4). Recently, two other isoforms, complexins 3 and 4, were identified; however, evidence of their specific function is unclear. Complexin 3 is expressed strongly in retina and certain regions of the brain including the hippocampus and thalamus, whereas complexin 4 is only expressed in retina (5).In neurons, the SNARE complex is a four-helical bundle composed of vesicle associated membrane protein (VAMP) 2 2, and the target SNAREs (t-SNAREs), syntaxin 1 and SNAP 25, located on the plasma membrane (6). Complexin 1 was orig...

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Section: Discussionsupporting

confidence: 69%

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Falkowski

Thomas

Groblewski

2010

Journal of Biological Chemistry

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“…As predicted by the structural data, the central ␣-helical region (aa 48 -70) was indispensable for the interaction with SNARE complexes (30,36,37). Site-specific mutations of the amino acids on CPX-I that are thought to contact the SNARE complex (R59H, R48A, R63A, and Y70A) showed a strong reduction in the binding capacity (Fig.…”

Section: Site-directed Mutagenesis Of Conserved Residues In Cpx-i -Imentioning

confidence: 94%

Distinct Domains of Complexins Bind SNARE Complexes and Clamp Fusion in Vitro

Giraudo

Garcia-Diaz

Eng

et al. 2008

Journal of Biological Chemistry

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In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca 2؉ ) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

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“…55 ). We found that the C 2 AB fragment displaces this complexin fragment from membrane-anchored SNARE complexes in the presence of Ca 2+ (ref.…”

Section: Structural Determinants Of Quaternary Sscap Complex Formationmentioning

confidence: 99%

A Quaternary SNARE–Synaptotagmin–Ca2+–Phospholipid Complex in Neurotransmitter Release

Han

Shen

Araç

et al. 2007

Journal of Molecular Biology

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203

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SummaryThe 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.

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Selective Interaction of Complexin with the Neuronal SNARE Complex

Cited by 169 publications

References 43 publications

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Distinct Domains of Complexins Bind SNARE Complexes and Clamp Fusion in Vitro

A Quaternary SNARE–Synaptotagmin–Ca2+–Phospholipid Complex in Neurotransmitter Release

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