A neuronal Sec1 homolog regulates neurotransmitter release at the squid giant synapse.
Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the squid giant synapse. Microinjection of s-Sec1 into the presynaptic terminal of the giant synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro, completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites ("active zones"), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin-at a stage blocked by the peptide-is necessary for docked vesicles to fuse.
Dresbach
T
T
Department of Neurochemistry, Max-Planck-Institute for Brain Research, 60528 Frankfurt, Germany.
Burns
M E
ME
O'Connor
V
V
DeBello
W M
WM
Betz
H
H
Augustine
G J
GJ
D000818:Animals; D003001:Cloning, Molecular; D049832:Decapodiformes / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008845:Microinjections; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000737:chemistry*; D009479:Neuropeptides / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins