Expression of synaptotagmin and syntaxin associated with N‐type calcium channels in small cell lung cancer

David, Pascale; Far, Oussama El; Martin-Mouto, Nicole; Takahashi, Masami; Seagar, Michael

doi:10.1016/0014-5793(93)81777-w

Cited by 42 publications

(13 citation statements)

References 24 publications

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“…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: 64%

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: Discussionmentioning

confidence: 64%

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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“…In addition to their interactions with syntaxin and SNAP-25, N-type Ca 2ϩ channels are coimmunoprecipitated with synaptotagmin (5,9,29,37,38), suggesting a possible interaction between them. Here we report that the synprint site of N-type Ca 2ϩ channels interacts with the second C2 domain (C2B) of synaptotagmin I (syt I) and competes with synaptotagmin for interaction with syntaxin in a Ca 2ϩ -dependent manner.…”

mentioning

confidence: 99%

Interaction of the synprint site of N-type Ca ²⁺ channels with the C2B domain of synaptotagmin I

Sheng

Yokoyama

Catterall

1997

Proc. Natl. Acad. Sci. U.S.A.

175

120

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N-type Ca 2؉ channels mediate Ca 2؉ inf lux, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their ␣ 1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca 2؉ channels with synaptotagmin I, an important Ca 2؉ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca 2؉ . Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca 2؉ -dependent because of the Ca 2؉ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca 2؉ channels and synaptotagmin, and suggest that N-type Ca 2؉ channels may undergo a complex series of Ca 2؉ -dependent interactions with multiple presynaptic proteins during neurotransmission.During synaptic transmission, exocytosis of synaptic vesicles in neurons is initiated by the rapid influx of Ca 2ϩ through voltage-gated Ca 2ϩ channels within 200 sec. Major progress has been made toward the understanding of the molecular mechanisms that underlie Ca 2ϩ -dependent exocytosis by identifying proteins that are involved in this process and analyzing their interactions (1, 2). Vesicle docking and fusion are mediated by a core complex of proteins including the synaptic vesicle protein VAMP͞synaptobrevin (3) and the plasmalemmal proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) (4-11). The synaptic vesicle protein p65 or synaptotagmin (12) binds Ca 2ϩ (13,14) and interacts with syntaxin in a Ca 2ϩ -dependent manner (15,16). It is thought to serve as a Ca 2ϩ sensor for fast, Ca 2ϩ -dependent neurotransmitter release (17)(18)(19)(20)(21)(22).N-type Ca 2ϩ channels are localized in nerve terminals (23-25) and participate in neurotransmitter release in central and peripheral synapses (26-28). They are coimmunoprecipitated and copurified with proteins of the synaptic core complex in immunochemical studies (4,5,29), and they are closely associated with sites of transmitter release in physiological experiments (30). The ␣ 1B subunits of N-type Ca 2ϩ channels (31, 32) interact with the synaptic core complex through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III (33, 34) in a Ca 2ϩ -dependent manner with maximum binding in the range of 10-30 M (35). Peptides containing th...

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“…Recently, the crystal structure of a soluble form of the complex formed between the v-SNARE synaptobrevin and the t-SNAREs syntaxin and SNAP-25 was solved, revealing a long four-helix coiled-coil as the basis of this stability. Synaptobrevin and the t-SNAREs syntaxin and SNAP-25 accumulate on different membranes, and a number of additional proteins have been identified that are capable of interacting with synaptobrevin, syntaxin, and SNAP-25 (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). These interactions may function to regulate the formation of the SNARE complex or the intracellular targeting of its constituents.…”

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confidence: 99%

Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

Skehel

Fabian-Fine

Kandel

2000

Proc. Natl. Acad. Sci. U.S.A.

130

113

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VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum-resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis.

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Expression of synaptotagmin and syntaxin associated with N‐type calcium channels in small cell lung cancer

Cited by 42 publications

References 24 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

Interaction of the synprint site of N-type Ca ²⁺ channels with the C2B domain of synaptotagmin I

Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

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Expression of synaptotagmin and syntaxin associated with N‐type calcium channels in small cell lung cancer

Cited by 42 publications

References 24 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

Interaction of the synprint site of N-type Ca 2+ channels with the C2B domain of synaptotagmin I

Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

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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

Interaction of the synprint site of N-type Ca ²⁺ channels with the C2B domain of synaptotagmin I