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.
Department of Chemistry and Biochemistry, Program in Biomolecular Science and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106-9510, USA.
Dawn T
DT
Bravo
Natalia G
NG
Kolmakova
Stanley M
SM
Parsons
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