Vesicle priming and recruitment by ubMunc13-2 are differentially regulated by calcium and calmodulin.
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.
Zikich
Dragoslav
D
Neurobiology Department, Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Mezer
Aviv
A
Varoqueaux
Frederique
F
Sheinin
Anton
A
Junge
Harald J
HJ
Nachliel
Esther
E
Melamed
Rely
R
Brose
Nils
N
Gutman
Menachem
M
Ashery
Uri
U
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*
D002147:Calmodulin; D009419:Nerve Tissue Proteins; C485548:UNC13B protein, human; D002118:Calcium