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

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.

Doc2b Ca2+-binding site mutants act as a gain of function at rest and loss of function during neuronal activity

“…In samples fixed at resting conditions, eGFP-tagged Doc2b WT was localized homogeneously throughout the cytosol similar to soluble eGFP, as expected for a cytosolic protein (Figure 2A) and in line with previous findings (Groffen et al, 2006). In contrast, eGFP-tagged Doc2b DN and Doc2b 6A both showed plasma membrane enrichment (Figure 2A), again consistent with previous studies (Friedrich et al, 2008; Houy et al, 2017).…”

“…Our data show that Doc2b 6A shows constitutive membrane binding, as supported by other studies (Houy et al, 2017; Courtney et al, 2018). Thus, Doc2b 6A is not a pure loss-of-function mutant.…”

“…In chromaffin granule secretion, wildtype Doc2b serves both positive and negative roles (Houy et al, 2017), a feature that is shared with other exocytotic proteins such as synaptotagmins, complexins and munc18s. In this system, Doc2b DN and Doc2b 6A favour immediate chromaffin granule fusion but impair sustained release at high [Ca 2+ ].…”

“…Consistently, Doc2 overexpression causes a Ca 2+ -independent, Munc-13 dependent release increase upon phorbol ester stimulation (Friedrich et al, 2013) and conversely, blockade of the Doc2-Munc13 interaction by synthetic peptides abolishes phorbol ester potentiation (Hori et al, 1999). Munc13-1 is necessary for the Doc2b-induced priming of secretory granules in chromaffin cells (Houy et al, 2017). However, alteration of this interaction by mutations in the MID domain have no effect on Ca 2+ -induced Doc2b migration to the membrane (Groffen et al, 2004; Gaffaney et al, 2014).…”

TMEM16A is indispensable for basal mucus secretion in airways and intestine

“…S6). Apical intracellular Ca 2+ prepares granules for release by the exocytic machinery via the Ca 2+ sensors Munc13 and double C2-like domain-containing protein beta (30)(31)(32). Thus, TMEM16A appears essential for exocytosis.…”