Distribution of Components of the SNARE Complex in Relation to Transmitter Release Sites at the Frog Neuromuscular Junction

Charvin, Nathalie; Boudier, Jean-Louis; Fathallah, Mohammed; Tagaya, Mitsuo; Takahashi, Masami; Seagar, Michael

doi:10.1111/j.1460-9568.1996.tb01239.x

Cited by 30 publications

(15 citation statements)

References 33 publications

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“…Immunocytochemical staining demonstrated that syntaxin 1 was consistently present in regions of high Ca channel staining. Previous studies have also noted a colocalization of syntaxin with the presynaptic Ca channel but that the distribution of syntaxin extends beyond that of the release sites (Bouvier et al, 1996;Raciborska et al, 1998). Our findings suggest that this syntaxin 1 is in a nonrelease site complex with MUNC18 -1.…”

Section: Discussionsupporting

confidence: 58%

“…Considerable evidence supports direct binding of these two proteins in vitro (Saisu et al, 1991;Yoshida et al, 1992;David et al, 1993;Sheng et al, 1994;Schmid et al, 1995;Vance et al, 1999) and their association has been demonstrated previously at the presynaptic terminal (Bouvier et al, 1996;Seagar and Takahashi, 1998). We first used Ab571 to confirm that syntaxin 1 coimmunoprecipitates with the N-type Ca channel in both chick brain and ciliary ganglion lysates (Fig.…”

Section: Discussionmentioning

confidence: 99%

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A Syntaxin 1, Gα_o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

Li¹,

Lau²,

Morris³

et al. 2004

J. Neurosci.

670

802

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Presynaptic Ca V 2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive G␣ O G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of Ca V 2.2 with syntaxin 1 and G␣ O . Ca V 2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with Ca V 2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the Ca V 2.2 puncta, whereas the bulk colocalized with MUNC18 -1. G␣ O, whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca V 2.2, MUNC18 -1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18 -1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with Ca V 2.2 and G␣ O , as well as in large clusters remote from the release site with MUNC18 -1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.

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Section: Discussionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

A Syntaxin 1, Gα_o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

Li¹,

Lau²,

Morris³

et al. 2004

J. Neurosci.

670

802

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“…This pattern indicates localization both in microvesicles docked at plasma membrane active zones and in clusters of microvesicles in the cytoplasm facing each active zone (Boudier et al 1996).…”

Section: Co-localization Of Calcium Channels Syntaxin 1 and Snap-25 mentioning

confidence: 88%

Interactions between proteins implicated in exocytosis and voltage–gated calcium channels

Seagar

Lévêque

Charvin

et al. 1999

Phil. Trans. R. Soc. Lond. B

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Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium in£ux through P/Q-type or N-type calcium channels. Puri¢cation of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N-and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immuno£uorescence confocal microscopy at the frog neuromuscular junction con¢rmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the a 1 A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35 S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the a 1 A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium in£ux close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.

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“…The timing of SNARE complex disassembly, and hence of NSF's action relative to the docking and fusion of two membranes has not been definitively established. In different systems, NSF appears to act before membrane docking (6,7), after membrane docking but before fusion (1,8), or even after fusion (9).…”

mentioning

confidence: 99%

A Revised Model for the Oligomeric State of the N-Ethylmaleimide-sensitive Fusion Protein, NSF

Fleming

Hohl

et al. 1998

Journal of Biological Chemistry

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The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s 20,w 0 , of 13.4 (؎0.1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.

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Distribution of Components of the SNARE Complex in Relation to Transmitter Release Sites at the Frog Neuromuscular Junction

Cited by 30 publications

References 33 publications

A Syntaxin 1, Gα_o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

A Syntaxin 1, Gα_o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

Interactions between proteins implicated in exocytosis and voltage–gated calcium channels

A Revised Model for the Oligomeric State of the N-Ethylmaleimide-sensitive Fusion Protein, NSF

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Distribution of Components of the SNARE Complex in Relation to Transmitter Release Sites at the Frog Neuromuscular Junction

Cited by 30 publications

References 33 publications

A Syntaxin 1, Gαo, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

A Syntaxin 1, Gαo, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

Interactions between proteins implicated in exocytosis and voltage–gated calcium channels

A Revised Model for the Oligomeric State of the N-Ethylmaleimide-sensitive Fusion Protein, NSF

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Product

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A Syntaxin 1, Gα_o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

A Syntaxin 1, Gα_o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization