Differential Regulation of Exocytosis by Calcium and CAPS in Semi-Intact Synaptosomes

Tandon, Anurag; Bannykh, Sergei I.; Kowalchyk, Judith A.; Banerjee, Abhijit; Martin, Thomas F.J.; Balch, William E.

doi:10.1016/s0896-6273(00)80522-x

Cited by 124 publications

(106 citation statements)

References 49 publications

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“…First, CAPS is present on dense-core but not synaptic vesicles in brain homogenates (8). Second, in functional assays with permeable brain synaptosomes, neutralizing CAPS antibodies interfere with Ca 2ϩ -triggered norepinephrine but not glutamate release (9). Consistent with a role in dense-core vesicle exocytosis, the present immunocytochemical studies localized CAPS to dense-core vesicles in melanotrophs (Fig.…”

Section: Discussionsupporting

confidence: 71%

“…CAPS is present on densecore but not synaptic vesicles in brain homogenates (8). Consistent with a distinct role in dense-core vesicle exocytosis, CAPS was found to be essential for Ca 2ϩ -dependent norepinephrine but not glutamate secretion in functional studies with permeable brain synaptosomes (9). In addition, the phenotype of loss-offunction mutants in Caenorhabditis elegans CAPS (UNC-31 protein) is distinct from that of mutants in other synaptic proteins such as synaptobrevin (10,11).…”

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confidence: 88%

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Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Rupnik

Kreft

Sikdar

et al. 2000

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

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Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca 2؉ -dependent activator protein for secretion), a protein required for Ca 2؉ -dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca 2؉ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca 2؉ dependencies. A threshold of Ϸ10 M Ca 2؉ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca 2؉ activity < 10 M. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca 2؉ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.capacitance ͉ pituitary cells ͉ rat melanotrophs

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Section: Discussionsupporting

confidence: 71%

mentioning

confidence: 88%

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Rupnik

Kreft

Sikdar

et al. 2000

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

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“…Synaptosome Preparation-Synaptosomes were prepared as described previously (17,18). Briefly, the murine brains minus cerebella were homogenized with 10 strokes of a Teflon pestle at 500 rpm in ice-cold buffer A (320 mM sucrose, 1 mM EGTA, and 5 mM HEPES, pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity*

Chen

Wislet

Samuel

et al. 2013

Journal of Biological Chemistry

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Background: ␣-Synuclein is known to undergo exchange between membrane and cytosolic compartments. Results: ␣-Synuclein interacts with GTP-bound Rab3a on synaptic vesicles, and its dissociation is mediated by GDI/Hsp90. Conclusion: ␣-Synuclein's membrane association and dissociation cycle is linked to synaptic activity by the Rab3a recycling machinery. Significance: Significance: Impairments to ␣-synuclein interactions with vesicles and with the Rab3a recycling machinery may affect neurodegeneration.

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“…Regulation of CAPS Dephosphorylation-CAPS is an abundant brain protein (2) that was shown to be required for Ca 2ϩ -triggered catecholamine secretion from brain synaptosome preparations (33,34). To determine whether the phosphorylation state of CAPS was altered during Ca 2ϩ -triggered exocytosis, we prepared synaptosomes for probing with the Ser(P)-5-and -6-specific CAPS antibody.…”

Section: Resultsmentioning

confidence: 99%

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

Nojiri

Loyet²,

Klenchin

et al. 2009

Journal of Biological Chemistry

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CAPS (Ca 2؉ -dependent activator protein for secretion) functions in priming Ca 2؉ -dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca 2؉ -dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca 2؉ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca 2ϩ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca 2ϩ -dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1 2 (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca 2ϩ -triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2-4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12)(13)(14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15,16). Several SNARE...

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Differential Regulation of Exocytosis by Calcium and CAPS in Semi-Intact Synaptosomes

Cited by 124 publications

References 49 publications

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity*

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

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