title: Titration of Syntaxin1 in mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability.
abstract: Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, NeuroCure Cluster of Excellence, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany, Department of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, AG Functional Cell Biology, Institute for Integrative Neuroanatomy, Charité Centre 2 for Basic Medicine, 10115 Berlin, Germany, and Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Marife
authors.initials: M
authors.lastname: Arancillo
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Min
authors.forename: Stefan
authors.initials: S
authors.lastname: Gerber
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Münster-Wandowski
authors.forename: Yuan-Ju
authors.initials: YJ
authors.lastname: Wu
authors.forename: Melissa
authors.initials: M
authors.lastname: Herman
authors.forename: Thorsten
authors.initials: T
authors.lastname: Trimbuch
authors.forename: Jong-Cheol
authors.initials: JC
authors.lastname: Rah
authors.forename: Gudrun
authors.initials: G
authors.lastname: Ahnert-Hilger
authors.forename: Dietmar
authors.initials: D
authors.lastname: Riedel
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Südhof
authors.forename: Christian
authors.initials: C
authors.lastname: Rosenmund
mesh_terms: D000818:Animals; D002460:Cell Line; D005089:Exocytosis / Q000502:physiology*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D013569:Synapses / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000235:genetics / Q000378:metabolism*; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D050827:Syntaxin 1