New transport assay demonstrates vesicular acetylcholine transporter has many alternative substrates.
The acetylcholine-binding site in vesicular acetylcholine transporter faces predominantly toward the outside of the vesicle when resting but predominantly toward the inside when transporting. Transport-related reorientation is detected by an ATP-induced decrease in the ability of saturating substrate to displace allosterically bound [(3)H]vesamicol. The assay was used here to determine whether structurally diverse compounds are transported by rat VAChT expressed in PC12(A123.7) cells. Competition by ethidium, tetraphenylphosphonium and other monovalent organic cations with [(3)H]vesamicol is decreased when ATP is added, and the effect depends on proton-motive force. The results indicate that many organic molecules carrying +1 charge are transported, even though the compounds do not resemble acetylcholine in structural details.
Bravo
Dawn T
DT
Department of Chemistry and Biochemistry, Program in Biomolecular Science and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106-9510, USA.
Kolmakova
Natalia G
NG
Parsons
Stanley M
SM
D000109:Acetylcholine / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology*; D001693:Biological Transport, Active / Q000502:physiology; D002412:Cations / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D015394:Molecular Structure; D009467:Neuromuscular Depolarizing Agents / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D016716:PC12 Cells; D010880:Piperidines / Q000378:metabolism; D051381:Rats; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism; D050494:Vesicular Acetylcholine Transport Proteins
D002412:Cations; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D010880:Piperidines; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; C006189:vesamicol; D000255:Adenosine Triphosphate; D004996:Ethidium; D000109:Acetylcholine; C013289:tetraphenylphosphonium