Dissociation of the vesicular acetylcholine transporter domains important for high-affinity transport recognition, binding of vesamicol and targeting to synaptic vesicles.
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.
Neuroscience Center, Louisiana State University Medical School, New Orleans 70112, USA.
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Varoqui
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Erickson
D000109:Acetylcholine / Q000378:metabolism; D000310:Adrenal Gland Neoplasms; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents / Q000493:pharmacokinetics; D009479:Neuropeptides; D016716:PC12 Cells; D010673:Pheochromocytoma; D010880:Piperidines / Q000493:pharmacokinetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013572:Synaptic Vesicles / Q000502:physiology*; D014162:Transfection; D050494:Vesicular Acetylcholine Transport Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents; D009479:Neuropeptides; D010880:Piperidines; D011993:Recombinant Fusion Proteins; C493459:SLC18A3 protein, human; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D033921:Vesicular Transport Proteins; C006189:vesamicol; D000109:Acetylcholine