Role of the polybasic sequence in the Doc2a C2B domain in dense-core vesicle exocytosis in PC12 cells

J. Neurochem. (2010) 114, 171-181.AbstractThe 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, Doc2a, Doc2b, and Doc2g, in humans and mice. Doc2a, 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 Doc2a (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 Doc2a(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.