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The Journal of Neuroscience, February 20, 2008, 28(8):1949-1960; doi:10.1523/JNEUROSCI.5096-07.2008

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Cellular/Molecular
Vesicle Priming and Recruitment by ubMunc13-2 Are Differentially Regulated by Calcium and Calmodulin

Dragoslav Zikich,^1 * Aviv Mezer,^2 * Frederique Varoqueaux,³ Anton Sheinin,¹ Harald J. Junge,³ Esther Nachliel,² Rely Melamed,¹ Nils Brose,³ Menachem Gutman,² and Uri Ashery¹

¹Neurobiology Department and ²Laser Laboratory for Fast Reactions in Biology, Department of Biochemistry, Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, and ³Department of Molecular Neurobiology, Max-Planck-Institute of Experimental Medicine, Göttingen 37075, Germany

Correspondence should be addressed to Uri Ashery, Department of Neurobiology, Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Email: uria{at}post.tau.ac.il

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.

Key words: Munc13; calmodulin; calcium; exocytosis; priming; chromaffin cell; kinetic modeling

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Received July 31, 2007; revised Dec. 20, 2007; accepted Dec. 24, 2007.

Correspondence should be addressed to Uri Ashery, Department of Neurobiology, Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Email: uria{at}post.tau.ac.il

This article has been cited by other articles:

				R. Friedrich, A. J. Groffen, E. Connell, J. R. T. van Weering, O. Gutman, Y. I. Henis, B. Davletov, and U. Ashery DOC2B Acts as a Calcium Switch and Enhances Vesicle Fusion J. Neurosci., July 2, 2008; 28(27): 6794 - 6806. [Abstract] [Full Text] [PDF]

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