title: Doc2b synchronizes secretion from chromaffin cells by stimulating fast and inhibiting sustained release.
abstract: Synaptotagmin-1 and -7 constitute the main calcium sensors mediating SNARE-dependent exocytosis in mouse chromaffin cells, but the role of a closely related calcium-binding protein, Doc2b, remains enigmatic. We investigated its role in chromaffin cells using Doc2b knock-out mice and high temporal resolution measurements of exocytosis. We found that the calcium dependence of vesicle priming and release triggering remained unchanged, ruling out an obligatory role for Doc2b in those processes. However, in the absence of Doc2b, release was shifted from the readily releasable pool to the subsequent sustained component. Conversely, upon overexpression of Doc2b, the sustained component was largely inhibited whereas the readily releasable pool was augmented. Electron microscopy revealed an increase in the total number of vesicles upon Doc2b overexpression, ruling out vesicle depletion as the cause for the reduced sustained component. Further experiments showed that, in the absence of Doc2b, the refilling of the readily releasable vesicle pools is faster, but incomplete. Faster refilling leads to an increase in the sustained component as newly primed vesicles fuse while the [Ca(2+)]i following stimulation is still high. We conclude that Doc2b acts to inhibit vesicle priming during prolonged calcium elevations, thus protecting unprimed vesicles from fusing prematurely, and redirecting them to refill the readily releasable pool after relaxation of the calcium signal. In sum, Doc2b favors fast, synchronized release, and limits out-of-phase secretion.
authors.affiliation: Department of Neuroscience and Pharmacology, Faculty of Health Sciences, and Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2200 Copenhagen N, Denmark, Center for Neurogenomics and Cognitive Research, Department of Functional Genomics, Neuroscience Campus Amsterdam, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands, and VU Medical Center, 1081 HV Amsterdam, The Netherlands.
authors.forename: Paulo S
authors.initials: PS
authors.lastname: Pinheiro
authors.forename: Heidi
authors.initials: H
authors.lastname: de Wit
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Walter
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Groffen
authors.forename: Matthijs
authors.initials: M
authors.lastname: Verhage
authors.forename: Jakob B
authors.initials: JB
authors.lastname: Sørensen
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism* / Q000648:ultrastructure; D005089:Exocytosis / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D050863:Synaptotagmin I / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; C490421:Doc2b protein, mouse; D009419:Nerve Tissue Proteins; D050863:Synaptotagmin I; D002118:Calcium