Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the alpha sub(4) beta sub(3) delta GABA sub(A) Receptor

• Published in: Molecular pharmacology Vol. 73; no. 3; pp. 960 - 967
• Main Authors: Barrera, Nelson P, Betts, Jill, You, Haitao, Henderson, Robert M, Martin, Ian L, Dunn, Susan MJ, Edwardson, JMichael
• Format: Journal Article
• Language: English
• Published: 01.03.2008
• ISSN: 0026-895X

The GABA sub(A) receptor is a chloride-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor consists of five subunits arranged pseudosymmetrically around a central pore. The predominant form of the receptor in the brain contains alpha sub(1)-, beta sub(2)-, and gamma sub(2)-subunits in the arrangement alpha beta alpha gamma beta , counter-clockwise around the pore. GABA sub(A) receptors containing delta -instead of gamma -subunits, although a minor component of the total receptor population, have interesting properties, such as an extrasynaptic location, high sensitivity to GABA, and potential association with conditions such as epilepsy. They are therefore attractive targets for drug development. Here we addressed the subunit arrangement within the alpha sub(4) beta sub(3) delta form of the receptor. Different epitope tags were engineered onto the three subunits, and complexes between receptors and anti-epitope antibodies were imaged by atomic force microscopy. Determination of the numbers of receptors doubly decorated by each of the three antibodies revealed a subunit stoichiometry of 2 alpha :2 beta :1 delta . The distributions of angles between pairs of antibodies against the alpha - and beta -subunits both had peaks at around 144 degree , indicating that these pairs of subunits were nonadjacent. Decoration of the receptor with ligands that bind to the extracellular domain (i.e., the lectin concanavalin A and an antibody that recognizes the beta -subunit N-terminal sequence) showed that the receptor preferentially binds to the mica extracellular face down. Given this orientation, the geometry of complexes of receptors with both an antibody against the delta -subunit and Fab fragments against the alpha -subunits indicates a predominant subunit arrangement of alpha beta alpha delta beta , counter-clockwise around the pore when viewed from the extracellular space.