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

Literature Life Science

Binding between subunits within an oligomeric cell surface receptor can be assessed using HTRF PPI reagents

Abstract

Binding between subunits within an oligomeric cell surface receptor can be assessed 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 alpga1beta2(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 XLL665 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, with 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)1and conclude that this method would have general applicability to other multisubunit cell surface proteins.

Details

J Biol Chem. 1999;274(15):10100-4.

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