Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer
| Autor: | Paul J. Whiting, Ruth M. McKernan, Sophie J. Farrar, Timothy P. Bonnert |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Flumazenil
Fluorophore Stereochemistry Ligands Biochemistry Ion Channels Cell Line Proto-Oncogene Proteins c-myc chemistry.chemical_compound Europium Cell surface receptor Organometallic Compounds Humans Receptor Molecular Biology Fluorescent Dyes Binding Sites GABAA receptor Antibodies Monoclonal Cell Biology Receptors GABA-A Acceptor Fluorescence Spectrometry Fluorescence Monomer Energy Transfer chemistry Ligand-gated ion channel Ion Channel Gating |
| Zdroj: | Journal of Biological Chemistry. 274:10100-10104 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.274.15.10100 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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