Differential surface accessibility of α(187–199) in the Torpedo acetylcholine receptor α subunits 1 1Edited by B. Holland

Fairclough, Robert H.; Twaddle, George M.; Gudipati, Eswari; Lin, Mike Y.; Richman, David P.

doi:10.1006/jmbi.1998.2001

Cited by 12 publications

(11 citation statements)

References 29 publications

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“…Other Abs that recognize ␣ subunits, such as the ␣ subunit-specific polyclonal Ab used in Figure 1 D, precipitate an excess of ␣ subunits relative to the other subunits at the early time points. Because mAb 383c binds to denatured ␣ subunits or linear stretches of the ␣ subunit virtually as well as it binds to fully assembled receptors (Fairclough et al, 1998b), the delayed recognition of ␣ subunits by mAb 383c indicates that the mAb 383c epitope is inaccessible at the early stages of AChR assembly. Thus, during AChR assembly, ␣ subunits appear to change conformation from a state in which residues 187-199 are inaccessible to mAb 383c to a state in which this region is accessible to the mAb.…”

Section: Resultsmentioning

confidence: 99%

“…The second ligand binding site on the AChR appears to differ structurally from the first site. The second ligand binding site, which forms at the ␣-␦ subunit interface, differs in its affinity for competitive antagonists (Neubig and Cohen, 1979;Sine and Taylor, 1981;Blount and Merlie, 1989;Pedersen and Cohen, 1990;Sine and Claudio, 1991) and is recognized by Abs different from those that recognize the first site (Mihovilovic and Richman, 1984;Dowding and Hall, 1987;Fairclough et al, 1998b). However, the same regions of the ␣ subunit and regions on the ␦ subunit analogous to those on the ␥ (or ⑀) subunit appear to contribute to the second site.…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

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

2001

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“…However, this idea was overturned by the observations that competitive antagonists, such as d -TC, bound with different affinities to the two binding sites and that the different affinities arose from intrinsic structural differences rather than from negative cooperativity (191, 250). Structural differences between the two binding sites were further demonstrated using the monoclonal antibody mAb 383C that targets residues 187–199 of the α -subunit (85). Rather than binding to both α -subunits, the antibody bound only to the α -subunit that forms the site with high affinity for d -TC.…”

Section: Structure-mechanism Relationshipsmentioning

confidence: 99%

End-Plate Acetylcholine Receptor: Structure, Mechanism, Pharmacology, and Disease

Sine

2012

Physiological Reviews

112

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The synapse is a localized neurohumoral contact between a neuron and an effector cell and may be considered the quantum of fast intercellular communication. Analogously, the postsynaptic neurotransmitter receptor may be considered the quantum of fast chemical to electrical transduction. Our understanding of postsynaptic receptors began to develop about a hundred years ago with the demonstration that electrical stimulation of the vagus nerve released acetylcholine and slowed the heart beat. During the past 50 years, advances in understanding postsynaptic receptors increased at a rapid pace, owing largely to studies of the acetylcholine receptor (AChR) at the motor endplate. The endplate AChR belongs to a large superfamily of neurotransmitter receptors, called Cys-loop receptors, and has served as an exemplar receptor for probing fundamental structures and mechanisms that underlie fast synaptic transmission in the central and peripheral nervous systems. Recent studies provide an increasingly detailed picture of the structure of the AChR and the symphony of molecular motions that underpin its remarkably fast and efficient chemoelectrical transduction.

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“…Note that the plateau of the β‐hairpin‐binding 383C is half the value of the plateau of the MIR‐directed 132A, suggesting 383C binds to only one of the two α subunit β‐hairpin loops. Further studies have marked the 383C‐binding α subunit β‐hairpin loop as one associated with the high‐affinity d ‐tubocurarine (dTC) binding site 8. Upon addition of carbamylcholine, the β‐hairpin loop epitope disappears (Fig.…”

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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Differential surface accessibility of α(187–199) in the Torpedo acetylcholine receptor α subunits 1 1Edited by B. Holland

Cited by 12 publications

References 29 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

End-Plate Acetylcholine Receptor: Structure, Mechanism, Pharmacology, and Disease

Agonist‐Induced Transitions of the Acetylcholine Receptor

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