Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.

Gershoni, Jonathan M.; Hawrot, Edward; Lentz, Thomas L.

doi:10.1073/pnas.80.16.4973

Cited by 108 publications

(67 citation statements)

References 23 publications

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“…The widely held concept that ␣-btx binds to the ␣ subunit can be traced to its ability to bind to isolated denatured ␣ subunits but not to ␤, ␥, or ␦ subunits (16). Also, within cells, ␣-btx binds to unassembled ␣ subunits but not to unassembled non-␣ subunits (17,18).…”

Section: Discussionmentioning

confidence: 99%

Identification of Equivalent Residues in the γ, δ, and ε Subunits of the Nicotinic Receptor That Contribute to α-Bungarotoxin Binding

Sine

1997

Journal of Biological Chemistry

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Cysteine was introduced from residues 116 to 121 of the ␥ subunit of the fetal mouse acetylcholine receptor, and the mutant receptors were treated with methanethiosulfonate reagents and examined for changes in ligand binding properties. Of the 18 combinations of mutant and reagent, only receptors harboring ␥L119C treated with the quaternary ammonium reagent MTSET (trimethylammonium-ethyl methanethiosulfonate) show a decreased number of ␣-bungarotoxin (␣-btx) sites. The decrease of 50% suggests that ␣-btx binding to the site harboring ␥L119C is blocked. Analysis of binding of the site-selective ligands dimethyl-d-tubocurarine (DMT) and ␣-conotoxin M1 (CTX) confirm specificity of modification for the site harboring ␥L119C. Cysteines placed at equivalent positions of the ␦ and ⑀ subunits also lead to selective loss of ␣-btx binding following MTSET treatment. ␥L119C receptors treated with the primary amine reagent MTSEA (aminoethyl methanethiosulfonate) retain ␣-btx binding to both sites but show reduced affinity for DMT and CTX at the modified site. Lysine mutagenesis of Leu ␥119 , Leu ␦121 , and Leu ⑀119 mimics MTSEA treatment, whereas mutagenesis of Thr ␣119 and Gln ␤119is without effect, demonstrating subunit and residue specificity of MTSEA modification. MTSET modification of nearby ␥Y117C does not block ␣-btx binding but markedly diminishes affinity for DMT and CTX. The overall findings indicate a localized point of interaction between ␣-btx and the modified ␥L119C, ␦L121C, and ⑀L119C.Acetylcholine receptors from vertebrate skeletal muscle are pentamers of homologous subunits with compositions ␣ 2 ␤␥␦ in fetal or ␣ 2 ␤⑀␦ in adult muscle (1). The functional receptor contains two acetylcholine binding sites, each generated by pairs of subunits, ␣␦ and either ␣␥ or ␣⑀ (2-4). Our current picture of the binding site is a subunit interface comprising three loops from the ␣ subunit and four loops from the non-␣ subunit (reviewed in Ref. 5). Although binding of small agonists and antagonists is mutually exclusive, their wide range in affinities for a given binding site likely owes to stabilization by different loops, whereas selectivity of a ligand for the three types of sites owes to differences in residues of a particular loop. Residues that stabilize small agonists and antagonists have been identified in both the ␣ and non-␣ subunits, yet residues that stabilize the much larger peptide antagonist ␣-bungarotoxin (␣-btx) 1 have been identified only in the ␣ subunit (6, 7).Photolabeling studies using either ␣-btx or an ␣-neurotoxin derivative demonstrated labeling of the ␥ and ␦ subunits of the Torpedo receptor, suggesting proximity of these subunits to the site of ␣-btx binding (8, 9). To identify portions of the non-␣ subunits close to the ␣-btx binding site, the present study substitutes cysteine into one of the four loops of the ␥ subunit that contribute to the binding site and examines ␣-btx binding following reaction with sulfhydryl-specific reagents. The siteselective ligands dimethyl-d-tubocurarine and ␣-conot...

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Section: Discussionmentioning

confidence: 99%

Identification of Equivalent Residues in the γ, δ, and ε Subunits of the Nicotinic Receptor That Contribute to α-Bungarotoxin Binding

Sine

1997

Journal of Biological Chemistry

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“…Several lines of evidence indicate that neighboring subunits affect ligand binding. a-BTX binds to isolated a subunit and not the other subunits (10,(16)(17)(18), while covalent incorporation of a-BTX by chemical (19) or photochemical (20) crosslinking indicates proximity of other subunits to the a-BTX binding site. Recent studies from the coexpression of pairs of subunits in quail fibroblasts indicate that coexpression of a and y subunits is required for highaffinity binding of TC, whereas low-affinity binding requires the coexpression ofa and 8 subunits (21).…”

mentioning

confidence: 99%

d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.

Pedersen

Cohen²

1990

Proc. Natl. Acad. Sci. U.S.A.

261

149

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“…The concentration of BTX that tion of_ 25I-BTX bindino to the isolated g-subunlt immobilized on filters was 1 x 10-7 M and 9 x 10 -M for d-tubocurare (18). Thus, t ie affinity of toxin binding to the 32-mer is as great as the affinity for the ntact but denatured g-subunit.…”

Section: Discussionmentioning

confidence: 81%

“…STX binds with lowered affinity to isolate!d a-subunit but not to B,y , and6 subunits (17)(18)(19). The competition between chol inergic agonists and neurotoxins suggests that the toxin-binding region of the a-subunit includes the ACh -binding site.…”

Section: Background and Review Of Literaturementioning

confidence: 99%

Neurotoxin-Binding Site on The Acetylcholine Receptor

Lentz

Wilson

1988

International Review of Neurobiology

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Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.

Cited by 108 publications

References 23 publications

Identification of Equivalent Residues in the γ, δ, and ε Subunits of the Nicotinic Receptor That Contribute to α-Bungarotoxin Binding

Identification of Equivalent Residues in the γ, δ, and ε Subunits of the Nicotinic Receptor That Contribute to α-Bungarotoxin Binding

d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.

Neurotoxin-Binding Site on The Acetylcholine Receptor

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