title: DOC2B acts as a calcium switch and enhances vesicle fusion.
abstract: Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.
authors.affiliation: Department of Neurobiology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Reut
authors.initials: R
authors.lastname: Friedrich
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Groffen
authors.forename: Emma
authors.initials: E
authors.lastname: Connell
authors.forename: Jan R T
authors.initials: JR
authors.lastname: van Weering
authors.forename: Orit
authors.initials: O
authors.lastname: Gutman
authors.forename: Yoav I
authors.initials: YI
authors.lastname: Henis
authors.forename: Bazbek
authors.initials: B
authors.lastname: Davletov
authors.forename: Uri
authors.initials: U
authors.lastname: Ashery
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002395:Catecholamines / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology*; D051379:Mice; D008813:Mice, Inbred ICR; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000235:genetics; D050600:SNARE Proteins / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D002135:Calcium-Binding Proteins; D002395:Catecholamines; C490421:Doc2b protein, mouse; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D002118:Calcium