Equilibrium binding and transport by vesicular acetylcholine transporter.
The method describes production and the selection of neurosecretory PC12A123.7 cells stably transfected with human vesicular acetylcholine transporter (hVAChT). Transfected cells provide postnuclear supernatant used to characterize equilibrium binding of the neurotransmitter acetylcholine (ACh), the pH dependence for transport of ACh, and the rate behavior for dissociation of the allosteric, high-affinity inhibitor vesamicol. Retention of radiolabeled ACh or vesamicol, mediated by hVAChT in synaptic-like microvesicles of postnuclear supernatant, is measured using filter assays. The procedure for regression analysis of data also is described.
Khare
Parul
P
Department of Chemistry and Biochemistry, Neuroscience Research Institute, University of California, Santa Barbara, CA, USA.
White
Aubrey R
AR
Mulakaluri
Anuprao
A
Parsons
Stanley M
SM
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D006863:Hydrogen-Ion Concentration; D016716:PC12 Cells; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D050494:Vesicular Acetylcholine Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D010880:Piperidines; D050494:Vesicular Acetylcholine Transport Proteins; C006189:vesamicol; D000109:Acetylcholine