Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1808, Issue 10, Pages 2435-2439Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2011.07.003
Keywords
Synaptotagmin; SNARE; Exocytosis; Liposome; Membrane fusion; Mast cell
Categories
Funding
- Astellas Foundation for Research on Medicinal Resources
- Ministry of Education, Culture, Sports, and Technology of Japan [16657046, 17049024]
- Nagoya City University
- Grants-in-Aid for Scientific Research [23590048, 17049024, 16657046] Funding Source: KAKEN
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Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca(2+) concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca(2+) dependent, the target molecule that interacts with Ca(2+) is not clear. Synaptotagmin is a Ca(2+) sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagrnin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca(2+), low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca(2+), and phosphatidylserine. (C) 2011 Elsevier B.V. All rights reserved.
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