Immobilized GABAA receptors and their ligand binding characteristics

Im, Wha Bin; Tai, Masaru; Blakeman, David P.; Davis, John P.

doi:10.1016/0006-291x(89)92181-5

Cited by 5 publications

(4 citation statements)

References 16 publications

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“…We then bound receptors to the two surfaces and incubated them with either concanavalin A, which should bind to the oligosaccharides on the extracellular face of the receptor (Im et al, 1989), or monoclonal antibody bd17, which recognizes an epitope at the N terminus of the ␤-subunit (Richards et al, 1987). We reasoned that changing the charge of the mica surface would change the predominant orientation of the receptors, depending on the charge distribution over the receptor surface.…”

Section: Resultsmentioning

confidence: 99%

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α₄β₃δ GABA_AReceptor

Barrera

Betts

You³

et al. 2007

Mol Pharmacol

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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 ␣ 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.

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Section: Resultsmentioning

confidence: 99%

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α₄β₃δ GABA_AReceptor

Barrera

Betts

You³

et al. 2007

Mol Pharmacol

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“…If this is indeed the case, then the orientation of the receptors should be reversed when they are bound to poly-L-glutamate-coated mica, which provides a negatively charged surface. To test these ideas, we bound receptors to the two surfaces and incubated them with either the lectin concanavalin A, which should bind to the oligosaccharides on the extracellular face of the receptor [33], or monoclonal antibody bd17, which recognises an epitope at the N terminus of the β-subunit [60]. We reasoned that receptors bound extracellular face down would have their binding sites for both concanavalin A and antibody bd17 occluded.…”

Section: Receptors Containing Three Types Of Subunit-determination Ofmentioning

confidence: 99%

Determination of the architecture of ionotropic receptors using AFM imaging

Barrera

Henderson

Edwardson

2007

Pflugers Arch - Eur J Physiol

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Fast neurotransmission in the nervous system is mediated by ionotropic receptors, all of which contain several subunits surrounding an integral ion channel. There are three major families of ionotropic receptors: the 'Cysloop' receptors (including the nicotinic receptor for acetylcholine, the 5-HT 3 receptor, the GABA A receptor and the glycine receptor), the glutamate receptors (including the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and N-methyl-D-aspartic acid receptors) and the P2X receptors for adenosine triphosphate. These receptors are often built from multiple types of subunit, raising the question of the stoichiometry and subunit arrangement within the receptors. This question is of therapeutic significance because in some cases drug-binding sites are located at subunit-subunit interfaces. In this paper, we describe a general method, based on atomic force microscopy imaging, to solve the architecture of multi-subunit proteins, such as the ionotropic receptors. Specific epitope tags are engineered onto each receptor subunit. The subunits are then expressed exogenously in cultured cells, and the receptors are isolated from detergent extracts of membrane fractions by affinity chromatography. The receptors are imaged both alone and in complex with anti-epitope antibodies. The size of the imaged particles provides an estimate of the subunit stoichiometry, whereas the geometry of the receptor-antibody complexes produces more detailed information about the receptor architecture. We use an automated, unbiased system to identify receptors and receptor-antibody complexes and to determine the geometry of the complexes. We are also able to determine the orientation of the receptors on the mica substrate, which will allow us to solve the subunit arrangement within receptors, such as the GABA A receptor, which contain three types of subunits.

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“…This result is particularly interesting because pregnanolone possess a 58 reduction moiety, and can also occur in brain tissue as a 5a analogue (Corpechot et al 1993). The 3a-hydroxyl moiety was also maintained to preserve the pregnane configuration which contributes to it hypnotic effects (Im et al 1989). During pregnancy, elevated plasma levels of progesterone directly correlates to increased concentrations of pregnanolone and 3a-hydroxy-5a-pregnan-20-one in the brain (Corpechot et al 1993).…”

Section: Tce and Steroid Metabolismmentioning

confidence: 95%

United States Air Force Summer Research Program -- 1993. Volume 7. Armstrong Laboratory

Jones¹,

Jabbour²,

Prasad³

et al. 1993

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Immobilized GABAA receptors and their ligand binding characteristics

Cited by 5 publications

References 16 publications

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α₄β₃δ GABA_AReceptor

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α₄β₃δ GABA_AReceptor

Determination of the architecture of ionotropic receptors using AFM imaging

United States Air Force Summer Research Program -- 1993. Volume 7. Armstrong Laboratory

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Immobilized GABAA receptors and their ligand binding characteristics

Cited by 5 publications

References 16 publications

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α4β3δ GABAAReceptor

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α4β3δ GABAAReceptor

Determination of the architecture of ionotropic receptors using AFM imaging

United States Air Force Summer Research Program -- 1993. Volume 7. Armstrong Laboratory

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Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α₄β₃δ GABA_AReceptor

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α₄β₃δ GABA_AReceptor