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 α1-, β2-, or γ2-subunits of the hetero-oligomeric γ- aminobutyric acid (GABA(A)) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2 (c-Myc), suggesting that there are 2x α-, 2x β-, and 1x γ-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 α1 (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 α1(c-Myc)β2γ2 and α1β2(c- Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABA(A) receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e. (α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.