Abstract
Chimeras between the human vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the human vesicular monoamine transporter (hVMAT2) have been constructed and stably expressed in a rat pheochromocytoma cell line (PC12) in an effort to identify cholinergic-specific domains of VAChT. Examination of the transport properties of a chimera in which the N-terminal portion (up to putative transmembrane domain II and including the lumenal glycosylated loop) of hVAChT was replaced with hVMAT2 sequences (2/V@NheI) revealed that its apparent affinity for acetylcholine (ACh) was reduced approximately seven-fold compared to wild-type. However, the affinity of this chimera for vesamicol did not significantly differ from hVAChT. Similarly, the 2/V@NheI chimera retained its preferential targeting to the small synaptic-like vesicles found in PC12 cells in agreement with our recently reported observations that the synaptic vesicle targeting domain resides in the cytoplasmic tail of VAChT.
Publication types
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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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Adrenal Gland Neoplasms
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Animals
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Binding Sites
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Biological Transport
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Carrier Proteins / chemistry*
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Carrier Proteins / metabolism*
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Humans
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Kinetics
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / metabolism*
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Membrane Transport Proteins*
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Neuromuscular Depolarizing Agents / pharmacokinetics
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Neuropeptides*
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PC12 Cells
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Pheochromocytoma
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Piperidines / pharmacokinetics*
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Rats
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Synaptic Vesicles / physiology*
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Transfection
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Vesicular Acetylcholine Transport Proteins
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Vesicular Biogenic Amine Transport Proteins
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Vesicular Monoamine Transport Proteins
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Vesicular Transport Proteins*
Substances
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Carrier Proteins
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Membrane Glycoproteins
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Membrane Transport Proteins
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Neuromuscular Depolarizing Agents
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Neuropeptides
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Piperidines
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Recombinant Fusion Proteins
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SLC18A3 protein, human
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Slc18a3 protein, rat
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Vesicular Acetylcholine Transport Proteins
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Vesicular Biogenic Amine Transport Proteins
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Vesicular Monoamine Transport Proteins
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Vesicular Transport Proteins
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vesamicol
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Acetylcholine