title: Vesicle priming and recruitment by ubMunc13-2 are differentially regulated by calcium and calmodulin.
abstract: Ca2+ regulates multiple processes in nerve terminals, including synaptic vesicle recruitment, priming, and fusion. Munc13s, the mammalian homologs of Caenorhabditis elegans Unc13, are essential vesicle-priming proteins and contain multiple regulatory domains that bind second messengers such as diacylglycerol and Ca2+/calmodulin (Ca2+/CaM). Binding of Ca2+/CaM is necessary for the regulatory effect that allows Munc13-1 and ubMunc13-2 to promote short-term synaptic plasticity. However, the relative contributions of Ca2+ and Ca2+/CaM to vesicle priming and recruitment by Munc13 are not known. Here, we investigated the effect of Ca2+/CaM binding on ubMunc13-2 activity in chromaffin cells via membrane-capacitance measurements and a detailed simulation of the exocytotic machinery. Stimulating secretion under various basal Ca2+ concentrations from cells overexpressing either ubMunc13-2 or a ubMunc13-2 mutant deficient in CaM binding enabled a distinction between the effects of Ca2+ and Ca2+/CaM. We show that vesicle priming by ubMunc13-2 is Ca2+ dependent but independent of CaM binding to ubMunc13-2. However, Ca2+/CaM binding to ubMunc13-2 specifically promotes vesicle recruitment during ongoing stimulation. Based on the experimental data and our simulation, we propose that ubMunc13-2 is activated by two Ca2+-dependent processes: a slow activation mode operating at low Ca2+ concentrations, in which ubMunc13-2 acts as a priming switch, and a fast mode at high Ca2+ concentrations, in which ubMunc13-2 is activated in a Ca2+/CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. These different Ca2+ activation steps determine the kinetic properties of exocytosis and vesicle recruitment and can thus alter plasticity and efficacy of transmitter release.
authors.affiliation: Neurobiology Department, Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Dragoslav
authors.initials: D
authors.lastname: Zikich
authors.forename: Aviv
authors.initials: A
authors.lastname: Mezer
authors.forename: Frederique
authors.initials: F
authors.lastname: Varoqueaux
authors.forename: Anton
authors.initials: A
authors.lastname: Sheinin
authors.forename: Harald J
authors.initials: HJ
authors.lastname: Junge
authors.forename: Esther
authors.initials: E
authors.lastname: Nachliel
authors.forename: Rely
authors.initials: R
authors.lastname: Melamed
authors.forename: Nils
authors.initials: N
authors.lastname: Brose
authors.forename: Menachem
authors.initials: M
authors.lastname: Gutman
authors.forename: Uri
authors.initials: U
authors.lastname: Ashery
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011999:Recruitment, Neurophysiological / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism / Q000502:physiology*
chemical_list: D002147:Calmodulin; D009419:Nerve Tissue Proteins; C485548:UNC13B protein, human; D002118:Calcium