Doc2b Is a High-Affinity Ca ²⁺ Sensor for Spontaneous Neurotransmitter Release

Abstract

Synaptic vesicle fusion in brain synapses occurs in phases that are either tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous) or as stochastic events in the absence of action potentials (spontaneous). Synaptotagmin-1, -2 and -9 are vesicleassociated Ca 2+ -sensors for synchronous release. Here we found that Double C2 domain (Doc2) proteins act as Ca 2+ -sensors to trigger spontaneous release. Although Doc2 proteins are cytosolic, they function analogously to synaptotagmin-1 but with a higher Ca 2+ -sensitivity and superior in vitro fusion-efficiency. Doc2 proteins bound to SNARE-complexes in competition with synaptotagmin-1. Thus, different classes of multiple C2 domain-containing molecules trigger synchronous versus spontaneous fusion, which suggests a general mechanism for synaptic vesicle fusion triggered by the combined actions of SNAREs and multiple C2 domain-containing proteins.Neurotransmitter release is triggered by a rise in intracellular Ca 2+ , which activates sensors that subsequently trigger vesicle fusion. Synchronous release, the fastest mode of neurotransmission, involves the Ca 2+ sensors synaptotagmin-1, -2 or -9 which are anchored in the vesicle membrane and contain two cytoplasmic C2 domains that bind phospholipids in a Ca 2+ -dependent manner and interact with the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (1-5). Synaptotagmin-1-deficient neurons lack synchronous release but display an increase in spontaneous release (6-9) except in autapses (1, 10), suggesting a distinct mechanism for spontaneous release. Spontaneous release occurs in the absence of action potentials and is largely Ca 2+ -dependent (12-16), although truly Ca 2+ -independent fusion may also exist (11). Doc2a and Doc2b are soluble proteins that contain C2 domains with high similarity to synaptotagmins (17). They are expressed in nerve terminals and interact with the secretory * To whom correspondence should be addressed. sander.groffen@cncr.vu.nl and sascha.martens@univie.ac.at.. 3 current address: Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9/3, Austria 8 These authors contributed equally to this work The authors declare no conflicting interests. Europe PMC Funders Group Role of Doc2b and Ca 2+ in spontaneous synaptic releaseWe generated Doc2b −/− mice by deleting the promoter and exon 1 of the Doc2b gene ( fig. S1) (23). Doc2b −/− mice did not express the remaining exons and lacked Doc2b immunoreactivity. Doc2b −/− mice were viable and fertile without gross abnormalities. Other proteins implicated in neurotransmitter secretion were expressed at normal levels ( fig. S1D).Compensatory ectopic expression of Doc2a was not detected in Doc2b-deficient brains by in situ hybridization and the Doc2a protein level was unchanged ( fig. S1). Doc2a −/− Doc2b −/− double knock-out (DKO) mice were also viable, fertile and indistinguishable with regard to gross anatomy. To study neurotransmission and synaptic plasticity ...

Structural insights into the Ca 2+ and PI(4,5)P 2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

“…S2C), making difficult to predict whether these domains might adopt the closed or open conformation without further experimentation. However, it is clear that the similarity of DOC2B to rabphilin 3A is higher than to syt1, and this might explain the different behaviors found for it in recent studies using KO mice (38)(39)(40).…”

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

“…Previous studies showed that multiple Ca 2+ sensors control spontaneous release, including Syt1 (15) and Doc2s (16,17). We therefore hypothesized that in the absence of Syt1 phosphorylation, Doc2s mediate the increased spontaneous release.…”

“…Syt1 is the vesicular Ca 2+ sensor that mediates fast AP-evoked release in the hippocampus (14) and drives a large fraction of spontaneous release (15). In the latter case, Syt1 competes with alternative sensors, in particular Doc2s, for SNARE binding and the initiation of vesicle fusion (16,17). Syt1 bears a highly conserved phosphorylation site (threonine 112) in a putative α-helix in the linker between the transmembrane (TM) and C2A domain that can be phosphorylated by PKC and Ca 2+ /calmodulin (CaM)-dependent protein kinase II (CaMK-II, refs.…”

Spontaneous transmitter release is critical for the induction of long-term and intermediate-term facilitation inAplysia

“…forms of release involve different Ca 2+ sensors (39). Compared with other members of the family, BoTx D has a greater effect on spontaneous release.…”