Abstract
Botulinum neurotoxins cleave synaptic SNAREs and block exocytosis, demonstrating that these proteins function in neurosecretion. However, the function of the SNARE syntaxin remains less clear because no neurotoxin cleaves it selectively. Starting with a botulinum neurotoxin that cleaves both syntaxin and SNAP-25, we engineered a version that retains activity against syntaxin but spares SNAP-25. These mutants block synaptic release in neurons and norepinephrine release in neuroendocrine cells, thus establishing an essential role for syntaxin in Ca2+-triggered exocytosis. These mutants can generate syntaxin-free cells as a useful experimental system for research and may lead to pharmaceuticals that target syntaxin selectively.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Blotting, Western
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Botulinum Toxins / genetics
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Botulinum Toxins / metabolism
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Calcium / metabolism
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Calcium / pharmacology
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Catalytic Domain
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Cells, Cultured
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Exocytosis / drug effects
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HEK293 Cells
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Humans
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Membrane Potentials
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Models, Molecular
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Mutation
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Neuroendocrine Cells / metabolism*
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Neurons / metabolism*
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Neurons / physiology
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Neurotoxins / chemistry
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Neurotoxins / genetics
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Neurotoxins / metabolism*
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Norepinephrine / metabolism
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PC12 Cells
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Patch-Clamp Techniques
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Protein Binding
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Protein Engineering
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Protein Structure, Tertiary
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Qa-SNARE Proteins / chemistry
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Qa-SNARE Proteins / genetics
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Qa-SNARE Proteins / metabolism*
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Rats
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Synaptosomal-Associated Protein 25 / chemistry
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Synaptosomal-Associated Protein 25 / genetics
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Synaptosomal-Associated Protein 25 / metabolism
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Transfection
Substances
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Neurotoxins
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Qa-SNARE Proteins
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Snap25 protein, rat
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Synaptosomal-Associated Protein 25
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Botulinum Toxins
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Calcium
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Norepinephrine