Mapping the mab 383C epitope to α 2 (187–199) of the Torpedo acetylcholine receptor on the three-dimensional model 1 1Edited by B. Holland

Fairclough, Robert H.; Twaddle, George M.; Gudipati, Eswari; Stone, Remington J.S.; Richman, David P.; Burkwall, David A.; Josephs, Robert

doi:10.1006/jmbi.1998.2000

Cited by 12 publications

(9 citation statements)

References 39 publications

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“…Later, the ratio of the ␣ to the other subunits almost doubles as ␣ 2 ␤␥␦ pentamers assemble (Green and Claudio, 1993). Because mAb 383c binds specifically to the 187-199 region (Fairclough et al, 1998a) and this region comprises a major part of the Bgt binding site, it appears that the 187-199 region becomes accessible to mAb 383c as part of the subunit folding that forms the first Bgt binding site. This interpretation of our data can explain why the Bgt binding site is not initially present after subunit synthesis, although Bgt can bind to the 173-204 peptide alone and to denatured ␣ subunits (Wilson et al, 1988;Chaturvedi et al, 1992).…”

Section: Resultsmentioning

confidence: 99%

“…The interaction between mAb 383c and Torpedo AChRs has been well characterized. Its epitope has been mapped to ␣ subunit residues 187-199 (Fairclough et al, 1998a), a region that contributes to both the Bgt and ACh binding sites. In support of this region contributing to the Bgt binding site, mAb 383c binding specifically inhibits Bgt binding to native Torpedo AChRs (Mihovilovic and Richman, 1987).…”

Section: Resultsmentioning

confidence: 99%

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Rearrangement of Nicotinic Receptor α Subunits during Formation of the Ligand Binding Sites

2001

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Muscle nicotinic acetylcholine receptors (AChRs) are pentamers that contain two ␣ subunits a ␤, ␥ (or ⑀), and ␦ subunit. In this paper, we have characterized subunit processing and folding events leading to formation of the two AChR ligand binding sites. ␣ subunit residues, 187-199, which are part of overlapping ACh and ␣-bungarotoxin (Bgt) binding sites on AChRs, were assayed using a monoclonal antibody (mAb) specific for these residues. We found that this region was inaccessible to the mAb early during AChR assembly but became accessible as the first of two Bgt binding sites formed later during assembly, indicating that the region changes conformation as the Bgt binding site appears. Without previous reduction, 20% of the ␣ subunits could be alkylated by bromoacetylcholine bromide as the first ACh binding site formed, which further indicated that the disulfide bond between cysteines 192 and 193 does not form until the first ACh binding site appears soon after Bgt binding site formation. When ␣ subunits were mutated to add a glycosylation site at residue 187, the number of Bgt binding sites increased threefold, AChRs assembled more efficiently, and 2.5-fold more AChRs reached the cell surface. Our results indicate that binding site formation involves a rate-limiting rearrangement of the ␣ subunit that exposes the 187-199 region to the endoplasmic reticulum lumen and determines when cysteines 192 and 193 disulfide bond.Key words: nicotinic receptor; Torpedo; muscle; ␣-bungarotoxin; protein folding and assembly; acetylcholine binding site Signal transduction by members of a family of neurotransmittergated ion channels, which include the acetylcholine (ACh), GABA A , glycine, and 5-HT 3 receptors, is initiated by binding of neurotransmitters to specific sites on the receptors. Muscle-type nicotinic ACh receptors (AChRs), which include the receptor in fish electric organs, have long served as a model for the family (for review, see Changeux, 1995;Karlin and Akabas, 1995). AChRs are composed of four homologous subunits, ␣, ␤, ␥ (or ⑀), and ␦, that assemble into pentamers with a stoichiometry of ␣ 2 ␤␥␦. There are two ligand binding sites per receptor. Affinity labeling and mutagenesis of the ␣ subunit have identified six residues, Tyr-93, , in the vicinity of the binding site and have shown that the adjacent cysteines, , are disulfide-bonded. Residues on the ␥ and ␦ subunits have also been identified as contributing to the binding site, from which it was concluded that the two binding sites are found near the interfaces between an ␣ subunit and either the ␥ or ␦ subunits (for review, see Karlin and Akabas, 1995;Tsigelny et al., 1997;Changeux and Edelstein, 1998).Venom-derived ␣-peptide neurotoxins are potent AChR competitive antagonists and bind with high affinity to regions overlapping ACh binding sites. The best characterized of these neurotoxins is ␣-bungarotoxin (Bgt), for which the major region contributing to the binding site has been localized to ␣ subunit residues 173-204 (Wilson et al., 1985;Tzartos and Rem...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Rearrangement of Nicotinic Receptor α Subunits during Formation of the Ligand Binding Sites

2001

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“…Anti‐AChR monoclonal antibody (mAb) 383C binds to the β‐hairpin loop, α(187‐199), of the Torpedo AChR 1. This loop is illustrated in Figure 1 (top) with a Cα trace highlighted on the gray Cα backbone of the acetylcholine binding protein (AChBP) of Brejc et al 2 The space‐filling side chains of Y190, C192/193, and Y198 are the four amino acids of this peptide that have been affinity‐labeled with cholinergic agonist and antagonist analogues 3‐7.…”

Section: Resultsmentioning

confidence: 99%

“…Figures 2, 7, and 8 are reprinted from references 8, 1, and 14, respectively, with permission from Elsevier. Figure 11 is reprinted from plate 1 found in reference 15.…”

Section: Acknowledgmentsmentioning

confidence: 99%

Agonist‐Induced Transitions of the Acetylcholine Receptor

Fairclough

Agius

Gudipati

et al. 2003

Annals of the New York Academy of Sciences

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Anti-acetylcholine receptor (AChR) monoclonal antibody 383C binds to the beta-hairpin loop alpha(187-199) of only one of the two Torpedo AChR alpha subunits. The loop recognized is associated with the alpha subunit corresponding to the high-affinity d-tubocurarine (dTC) binding site. Desensitization of the receptor with carbamylcholine completely blocks the binding of 383C. Mild reduction of AChR alpha subunit cys 192-193 disulfide with DTT and subsequent reaction with 5-iodoacetamidofluorescein label only the high-affinity dTC alpha subunit. Rhodamine-labeled alpha-bungarotoxin (R-Btx) binds to the unlabeled AChR alpha subunit as monitored by fluorescence resonance energy transfer between the fluorescein and rhodamine dyes. A 10-A contraction of the distance between the dyes is observed following the addition of carbamylcholine. In a small angle X-ray diffraction experiment exploiting anomalous X-ray scattering from Tb(III) ions titrated into AChR Ca(II) binding sites, we find evidence for a change in the Tb(III) ion distribution in the region of the ion channel following addition of carbamylcholine to the AChR. The carbamylcholine-induced loss of the 383C epitope, the 10-A contraction of the beta-hairpin loop, and the loss of multivalent cations from the channel likely represent the first molecular transitions leading to AChR channel opening.

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“…Given the central role of the residues of this peptide in binding the neurotransmitter as well as 383C, we have mapped the combining site of 383C onto the 3-D model of the AChR via small-angle X-ray diffraction of AChR-enriched membrane vesicles decorated with 383C along with image processing of electron micrographs of tubular crystalline arrays of AChR decorated with 383C. 1 The image analysis reveals a single decorated ␣-subunit on the AChR; 1 this is confirmed by measuring the 383C binding stoichiometry to AChR-enriched membrane vesicles, which is one 383C/two toxin sites. 8 The particular ␣-subunit decorated by 383C is the ␣ 2 -subunit 1 located between ␤ and ␥ in the scheme of Kubalek et al 9 and is illustrated in FIGURE 2a.…”

Section: Location Of 383c Epitope On the Achr Modelmentioning

confidence: 99%

A Role for α(187–199) in the Conversion of Agonist Binding Energy to the Opening of the Acetylcholine Receptor Ion Channel^a

Fairclough

Gudipati

Lin

et al. 1998

Annals of the New York Academy of Sciences

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Mapping the mab 383C epitope to α 2 (187–199) of the Torpedo acetylcholine receptor on the three-dimensional model 1 1Edited by B. Holland

Cited by 12 publications

References 39 publications

Rearrangement of Nicotinic Receptor α Subunits during Formation of the Ligand Binding Sites

Rearrangement of Nicotinic Receptor α Subunits during Formation of the Ligand Binding Sites

Agonist‐Induced Transitions of the Acetylcholine Receptor

A Role for α(187–199) in the Conversion of Agonist Binding Energy to the Opening of the Acetylcholine Receptor Ion Channel^a

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Mapping the mab 383C epitope to α 2 (187–199) of the Torpedo acetylcholine receptor on the three-dimensional model 1 1Edited by B. Holland

Cited by 12 publications

References 39 publications

Rearrangement of Nicotinic Receptor α Subunits during Formation of the Ligand Binding Sites

Rearrangement of Nicotinic Receptor α Subunits during Formation of the Ligand Binding Sites

Agonist‐Induced Transitions of the Acetylcholine Receptor

A Role for α(187–199) in the Conversion of Agonist Binding Energy to the Opening of the Acetylcholine Receptor Ion Channela

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A Role for α(187–199) in the Conversion of Agonist Binding Energy to the Opening of the Acetylcholine Receptor Ion Channel^a