Received for publication October 4, 2007.
Revised December 11, 2007.
Accepted for publication December 12, 2007.

Atomic force microscopy reveals the stoichiometry and subunit arrangement of the ₄₃ GABA_A receptor

Abstract

The GABA_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 ₁-, ₂-, and ₂-subunits in the arrangement , counter-clockwise around the pore. GABA_A 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 ₄₃ 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 non-adjacent. 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.

Key words: GABAA, GABAC, Structure determinations

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