title: Radioligand binding to nanodisc-reconstituted membrane transporters assessed by the scintillation proximity assay.
abstract: The scintillation proximity assay is a powerful technique for measuring radioligand binding to membrane transporters and has become an integral part of high-throughput drug discovery screening efforts. Here we adapt the method for use with purified LeuT, a prokaryotic secondary transporter, reconstituted into phospholipid bilayer nanodiscs. This application surmounts potential challenges with background interference from endogenously expressed proteins, aggregation and loss of binding activity often accompanying detergent solubilization from native cell membranes, and heterogeneity in size and transporter orientation, where at least some ligand binding sites are inaccessible, associated with reconstitution into lipid vesicles.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
authors.forename: Mahmoud L
authors.initials: ML
authors.lastname: Nasr
authors.forename: Satinder K
authors.initials: SK
authors.lastname: Singh
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000032:analysis* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism*; D008024:Ligands; D008051:Lipid Bilayers / Q000378:metabolism; D049329:Nanostructures / Q000737:chemistry; D012588:Scintillation Counting
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins; D008024:Ligands; D008051:Lipid Bilayers