期刊
JOURNAL OF NEUROCHEMISTRY
卷 114, 期 1, 页码 171-181出版社
WILEY-BLACKWELL
DOI: 10.1111/j.1471-4159.2010.06739.x
关键词
C2 domain; dense-core vesicle; Doc2; exocytosis; SNAP-25; syntaxin-1a
资金
- Ministry of Education, Culture, Sports, and Technology (MEXT) of Japan [18689008, 21790197, 20113006, 21370087]
- Naito Foundation
- Research Foundation for Opto-Science and Technology
- Nakajima Foundation
- Grants-in-Aid for Scientific Research [18689008, 21370087, 21790197, 20113006] Funding Source: KAKEN
P>The double C2 (Doc2) family is characterized by an N-terminal Munc13-1-interacting domain and C-terminal tandem C2 domains, and it comprises three isoforms, Doc2 alpha, Doc2 beta, and Doc2 gamma, in humans and mice. Doc2 alpha, the best-characterized, brain-specific isoform, exhibits Ca2+-dependent phospholipid-binding activity through its C2A domain, and the Ca2+-binding activity is thought to be important for the regulation of Ca2+-dependent exocytosis. In contrast to the C2A domain, however, nothing is known about the physiological functions of the C2B domain in regulated exocytosis. In this study, we demonstrated by a mutation analysis that the polybasic sequence in the C2B domain of Doc2 alpha (306 KKSKHKTCVKKK 317) is required for binding of syntaxin-1a/synaptosome-associated protein of 25 kDa (SNAP-25) heterodimer. We also investigated the effect of Lys-to-Gln (named KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in PC12 cells. A Doc2 alpha(KQ) mutant, which lacks binding activity toward syntaxin-1a/SNAP-25 heterodimer, significantly decreased the number of plasma membrane-docked vesicles before stimulation and strongly inhibited high-KCl-induced exocytosis from the plasma membrane-docked vesicles. These results indicate that the polybasic sequence in the C2B domain functions as a binding site for syntaxin-1a/SNAP-25 heterodimer and controls the number of 'readily releasable' vesicles in neuroendocrine cells.
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