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 ...

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

No abstract