Abstract
A putative vesicular acetylcholine transporter (VAChT) was overexpressed in developing Xenopus spinal neurons by injection of rat VAChT cDNA or synthetic mRNA into Xenopus embryos. This resulted in a marked increase in the amplitude and frequency of miniature excitatory postsynaptic currents at neuromuscular synapses, reflecting an over 10-fold increase in the vesicular packaging of acetylcholine (ACh). The effect appeared in developing neurons even before synaptogenesis and was blocked by L-vesamicol, a specific blocker of ACh uptake into synaptic vesicles. Mutational studies showed that two highly conserved aspartate residues within putative transmembrane domains 4 and 10 are essential for the transport activity. These results provide direct evidence for the physiological function of a putative VAChT and demonstrate that quantal size can be regulated by changes in vesicular transporter activity.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Acetylcholine / metabolism*
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Animals
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Aspartic Acid / genetics
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Blastomeres
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Carrier Proteins / analysis
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Carrier Proteins / genetics*
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Carrier Proteins / metabolism
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Cells, Cultured
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Embryo, Nonmammalian / chemistry
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Embryo, Nonmammalian / physiology
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Female
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Gene Expression Regulation, Developmental / physiology
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Membrane Transport Proteins*
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Microinjections
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Muscles / cytology
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Mutagenesis / physiology
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Nervous System / cytology
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Neuromuscular Depolarizing Agents / pharmacology
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Neurons / chemistry
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Neurons / physiology
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Neurons / ultrastructure
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Patch-Clamp Techniques
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Piperidines / pharmacology
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Rats
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Synapses / chemistry
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Synapses / physiology
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Synaptic Transmission / drug effects
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Synaptic Transmission / physiology*
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Synaptophysin / analysis
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Vesicular Acetylcholine Transport Proteins
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Vesicular Transport Proteins*
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Xenopus laevis
Substances
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Carrier Proteins
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Membrane Transport Proteins
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Neuromuscular Depolarizing Agents
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Piperidines
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Slc18a3 protein, rat
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Synaptophysin
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Vesicular Acetylcholine Transport Proteins
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Vesicular Transport Proteins
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Aspartic Acid
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vesamicol
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Acetylcholine