Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer

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 alpha(1)-, beta(2)-, or gamma(2)-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABA(A)) receptor in intact cells. The maximal fluorescent signal derived from the alpha(1)(c-Myc)beta(2)gamma(2) and the alpha(1)beta(2)(c-Myc)gamma(2) receptors was twice that obtained with alpha(1)beta(2)gamma(2)(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 alpha(1)(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 alpha(1)(c-Myc)beta(2)gamma(2) and alpha(1)beta(2)(c-Myc)gamma(2), but no significant energy transfer was observed with alpha(1)beta(2)gamma(2)(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer, We confirm that the GABA, receptor subtype, alpha(1)beta(2)gamma(2), is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i,e, (alpha(1)),(beta(2))(2)(gamma(2))(1)) and conclude that this method would have general applicability to other multisubunit cell surface proteins.