title: Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer.
abstract: We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.
authors: Department of Biochemistry and Molecular Biology, Merck Sharp and Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, United Kingdom.
authors: S J
authors: SJ
authors: Farrar
authors: P J
authors: PJ
authors: Whiting
authors: T P
authors: TP
authors: Bonnert
authors: R M
authors: RM
authors: McKernan
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D004735:Energy Transfer; D005063:Europium; D005442:Flumazenil / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology*; D008024:Ligands; D009942:Organometallic Compounds; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology; D011963:Receptors, GABA-A / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007473:Ion Channels; D008024:Ligands; D009942:Organometallic Compounds; D016271:Proto-Oncogene Proteins c-myc; D011963:Receptors, GABA-A; C070734:europium(III) trisbipyridine cryptate; D005442:Flumazenil; D005063:Europium