Differential properties of GTP- and Ca(2+)-stimulated exocytosis from large dense core vesicles.
Many cells utilize a GTP-dependent pathway to trigger exocytosis in addition to Ca(2+)-triggered exocytosis. However, little is known about the mechanism by which GTP triggers exocytosis independent of Ca(2+). We used dual-color evanescent field microscopy to compare the motion and fusion of large dense core vesicles stimulated by either mastoparan (Mas) in Ca(2+)-free conditions or high K(+) in the presence of Ca(2+). We demonstrate that Mas is hardly effective in triggering the fusion of the predocked vesicles but predominantly mobilizes cytosolic vesicles. In contrast, Ca(2+)-dependent exocytosis is largely due to predocked vesicles. Fusion kinetics analysis and carbon-fiber amperometry reveal that Mas induces a brief 'kiss-and-run' fusion and releases only a small amount of the cargo, whereas Ca(2+) stimulates a more persistent opening of the fusion pore and larger release of the contents. Furthermore, we show that Mas-released vesicles require a much shorter time to reach fusion competence once they approach the plasma membrane. Our data suggest the involvement of different mechanisms not only in triggering and fusion but also in the docking and priming process for Ca(2+)- and GTP-dependent exocytosis.
Bai
Li
L
Joint Laboratory of Institute of Biophysics and Huazhong University of Science and Technology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Zhu
Dan
D
Zhou
Keming
K
Zhou
Wei
W
Li
Dongdong
D
Wang
Yan
Y
Zhang
Rongying
R
Xu
Tao
T
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D008561:Membrane Fusion / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods*; D016716:PC12 Cells; D010455:Peptides / Q000378:metabolism; D010777:Photochemistry; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D014862:Wasp Venoms / Q000378:metabolism
D005456:Fluorescent Dyes; D036341:Intercellular Signaling Peptides and Proteins; C485187:PHluorin; D010455:Peptides; D050988:Vesicle-Associated Membrane Protein 2; D014862:Wasp Venoms; D049452:Green Fluorescent Proteins; C037500:mastoparan; D006160:Guanosine Triphosphate; D002118:Calcium