Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the 4 3 GABAA Receptor

The GABAA 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 1-, 2-, and 2-subunits in the arrangement , counter-clockwise around the pore. GABAA receptors containing instead of -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 4 3 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 :2 :1 . The distributions of angles between pairs of antibodies against the and -subunits both had peaks at around 144°, 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 -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 -subunit and Fab fragments against the -subunits indicates a predominant subunit arrangement of , counter-clockwise around the pore when viewed from the extracellular space. The GABAA receptor, responsible for fast inhibitory transmission in the central nervous system, is a member of the Cys-loop ligand-gated ion channel superfamily, together with the nicotinic acetylcholine receptor, the 5-HT3 receptor, and the glycine receptor (Karlin, 2002; Lester et al., 2004). The receptor exists as a heteromeric complex of five subunits, arranged pseudo-symmetrically around a central Cl ion channel (Sieghart, 1995). Electron microscopy of samples of purified GABAA receptor reveals a cylinder of external diameter 7 nm with a central pore of diameter 2 to 3 nm (Nayeem et al., 1994). Nineteen GABAA receptor subunit isoforms have so far been identified (Barnard et al., 1998). The predominant form of the receptor in the brain contains 1-, 2-, and 2-subunits in the stoichiometry 2 :2 :1 (Farrar et al., 1999). Previous work, in which various combinations of concatenated subunits were expressed in Xenopus laevis oocytes, indicated a subunit arrangement of , reading counterclockwise around the pore when viewed from the extracellular face of the membrane (Baumann et al., 2002; Baur et al.,