title: C-terminal complexin sequence is selectively required for clamping and priming but not for Ca2+ triggering of synaptic exocytosis.
abstract: Complexins are small soluble proteins that bind to assembling SNARE complexes during synaptic vesicle exocytosis, which in turn mediates neurotransmitter release. Complexins are required for clamping of spontaneous "mini " release and for the priming and synaptotagmin-dependent Ca(2+) triggering of evoked release. Mammalian genomes encode four complexins that are composed of an N-terminal unstructured sequence that activates synaptic exocytosis, an accessory α-helix that clamps exocytosis, an essential central α-helix that binds to assembling SNARE complexes and is required for all of its functions, and a long, apparently unstructured C-terminal sequence whose function remains unclear. Here, we used cultured mouse neurons to show that the C-terminal sequence of complexin-1 is not required for its synaptotagmin-activating function but is essential for its priming and clamping functions. Wild-type complexin-3 did not clamp exocytosis but nevertheless fully primed and activated exocytosis. Strikingly, exchanging the complexin-1 C terminus for the complexin-3 C terminus abrogated clamping, whereas exchanging the complexin-3 C terminus for the complexin-1 C terminus enabled clamping. Analysis of point mutations in the complexin-1 C terminus identified two single amino-acid substitutions that impaired clamping without altering the activation function of complexin-1. Examination of release induced by stimulus trains revealed that clamping-deficient C-terminal complexin mutants produced a modest relative increase in delayed release. Overall, our results show that the relatively large C-terminal complexin-1 sequence acts in priming and clamping synaptic exocytosis and demonstrate that the clamping function is not conserved in complexin-3, presumably because of its distinct C-terminal sequences.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, CA 94305-5453, USA.
authors.forename: Yea Jin
authors.initials: YJ
authors.lastname: Kaeser-Woo
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Yang
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Südhof
mesh_terms: D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D055592:Biophysical Phenomena / Q000187:drug effects / Q000235:genetics; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004558:Electric Stimulation; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology*; D018408:Patch-Clamp Techniques; D017354:Point Mutation / Q000502:physiology; D017433:Protein Structure, Secondary / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D013569:Synapses / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D013572:Synaptic Vesicles / Q000187:drug effects / Q000502:physiology; D050857:Synaptotagmins; D014161:Transduction, Genetic / Q000379:methods
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C529624:Cplx3 protein, mouse; D018691:Excitatory Amino Acid Antagonists; D005136:Eye Proteins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D050600:SNARE Proteins; C095992:complexin I; D050857:Synaptotagmins; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D002118:Calcium