Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs

Abstract
Article and author information
Metrics

Abstract

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca²⁺ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca²⁺-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca²⁺-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca²⁺-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping protein domain (the MID-domain), but is separate from its Ca²⁺-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.

Article and author information

Author details

Sébastien Houy

Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark

Competing interests
No competing interests declared.
Alexander J Groffen

Department of Functional Genomics, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

Competing interests
No competing interests declared.
Iwona Ziomkiewicz

Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark

Competing interests
Iwona Ziomkiewicz, Performed experiments as an employee of University of Copenhagen and is now an employee of AstraZeneca. Has no financial investments in AstraZeneca.
Matthijs Verhage

Department of Functional Genomics, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

Competing interests
No competing interests declared.
Paulo S Pinheiro

Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark

Competing interests
No competing interests declared.
Jakob Balslev Sørensen

Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark

For correspondence
jakobbs@sund.ku.dk

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-5465-3769

Funding

Lundbeckfonden

Jakob Balslev Sørensen

Novo Nordisk Foundation

Jakob Balslev Sørensen

Danish Medical Research Council

Sébastien Houy

European Research Council (ERC-ADG-322966-DCVfusion)

Matthijs Verhage

Danish Medical Research Council

Jakob Balslev Sørensen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Permission to keep and breed knockout mice for this study was obtained fromThe Danish Animal Experiments Inspectorate (2006/562−43, 2012−15−2935−00001). The animals were maintained in an AAALAC-accredited stable in accordance with institutional guidelines as overseenby the Institutional Animal Care and Use Committee (IACUC).

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.