Tomosyn inhibits synaptotagmin-1-mediated step of Ca2+-dependent neurotransmitter release through its N-terminal WD40 repeats.
Neurotransmitter release is triggered by Ca(2+) binding to a low affinity Ca(2+) sensor, mostly synaptotagmin-1, which catalyzes SNARE-mediated synaptic vesicle fusion. Tomosyn negatively regulates Ca(2+)-dependent neurotransmitter release by sequestering target SNAREs through the C-terminal VAMP-like domain. In addition to the C terminus, the N-terminal WD40 repeats of tomosyn also have potent inhibitory activity toward Ca(2+)-dependent neurotransmitter release, although the molecular mechanism underlying this effect remains elusive. Here, we show that through its N-terminal WD40 repeats tomosyn directly binds to synaptotagmin-1 in a Ca(2+)-dependent manner. The N-terminal WD40 repeats impaired the activities of synaptotagmin-1 to promote SNARE complex-mediated membrane fusion and to bend the lipid bilayers. Decreased acetylcholine release from N-terminal WD40 repeat-microinjected superior cervical ganglion neurons was relieved by microinjection of the cytoplasmic domain of synaptotagmin-1. These results indicate that, upon direct binding, the N-terminal WD40 repeats negatively regulate the synaptotagmin-1-mediated step of Ca(2+)-dependent neurotransmitter release. Furthermore, we show that synaptotagmin-1 binding enhances the target SNARE-sequestering activity of tomosyn. These results suggest that the interplay between tomosyn and synaptotagmin-1 underlies inhibitory control of Ca(2+)-dependent neurotransmitter release.
Division of Membrane Dynamics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Yasunori
Y
Yamamoto
Sumiko
S
Mochida
Naoyuki
N
Miyazaki
Katsuhisa
K
Kawai
Kohei
K
Fujikura
Takao
T
Kurooka
Kenji
K
Iwasaki
Toshiaki
T
Sakisaka
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000378:metabolism*; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C484769:Stxbp5 protein, rat; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D002118:Calcium