Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by .alpha.-neurotoxin crosslinking

Hamilton, Susan L.; Pratt, David R.; Eaton, Douglas C.

doi:10.1021/bi00330a015

Cited by 40 publications

(26 citation statements)

References 37 publications

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“…a-Btx has been shown to bind to the a subunits (22,23), and since the binding of a-Btx and a-Ntx is mutually exclusive and they both inhibit photoaffinity labeling of the a-subunit (24,25), it is believed that also a-Ntx binds to the high-affinity binding sites for cholinergic ligands present on the a subunits. Results, obtained with a-Ntx cross-linked to the AcChoR subunits and with an electron microscopic approach using a-Ntx complexed to biotin-avidin, support the notion that also a-Ntx binds to the a subunits (26,27). a-Ntx and a-Btx bind to the AcChoR molecule with a stoichiometry of 2:1 (reviewed in ref.…”

mentioning

confidence: 56%

“…a-Dtx is a cholinergic antagonist closely related to a-Ntx and a-Btx because it belongs to the same protein family and is highly homologous to them (21,39). These toxins are powerful blockers of muscle and electric organ AcChoR by virtue of their ability to bind to the two a subunits (22,23,26,27) in a competitive fashion with acetylcholine, carbamoylcholine, curare, and other classic cholinergic ligands (21,52). a-Btx binds to a segment of the a subunit very close to or overlapping with the segment containing a cysteine residue labeled by cholinergic labels (53).…”

Section: Effect Of A-dtx On the Dissociation Rate Of A -Btx-acchormentioning

confidence: 99%

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Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

Conti‐Tronconi¹,

Raftery²

1986

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

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We have studied the stoichiometry of the binding of the long a-neurotoxins from the venom of Dendroaspis viridis (a-dendrotoxin) and Naja naja siamensis (a-cobratoxin) to the membrane-bound acetylcholine receptor (AcChoR) from Torpedo californica electric organ. The number of toxin molecules bound to one AcChoR molecule was determined by simultaneous-quantitative gas-phase microsequencing of all the amino acid sequences present in AcChoR-aneurotoxin complexes. This method permits the use of homogeneous (nonradiolabeled) preparations of native toxins to obtain molar ratios of neurotoxin-receptor complexes. The stoichiometry obtained for a-cobratoxin was 2.1 ± 0.2 (n = 4), in agreement with the accepted view that a-cobratoxin, like a-bungarotoxin, binds to the two a subunits, which are constituent polypeptides of the AcChoR molecule. aDendrotoxin gave a stoichiometry of 4.1 ± 0.5 (n = 12); therefore, the AcChoR molecule contains four binding sites for this a-neurotoxin, two of which are recognized by acobratoxin. In support of this contention we have also found that when the AcChoR is saturated with a-bungarotoxin, addition of a-dendrotoxin markedly accelerates the dissociation of the bound a-bungarotoxin, demonstrating that the occupancy of the additional two sites by the latter toxin influences and decreases the affinity of the former toxin for its two binding sites. The fact that the AcChoR molecule is a pseudosymmetric complex of five highly homologous peptides suggests the possibility that as many as five binding sites for cholinergic ligand could be present, one on each subunit.The nicotinic acetylcholine receptor (AcChoR) from Torpedo electric organ is a complex pentameric molecule formed by four highly homologous proteins in a stoichiometry a2Pfy8 (reviewed in ref.

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mentioning

confidence: 56%

Section: Effect Of A-dtx On the Dissociation Rate Of A -Btx-acchormentioning

confidence: 99%

Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

Conti‐Tronconi¹,

Raftery²

1986

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

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“…However, it was noted (8) that p-(dimethylamino)benzenediazonium fluoroborate was specifically photoincorporated into the y subunit at a level 10%1 of a subunit. In addition, chemical crosslinking of 3H-labeled cobra toxin to the AcChoR resulted in reaction with all subunits that could be blocked selectively by TC (19). UV-induced crosslinking of '25I-labeled a-BTX to the AcChoR resulted in covalent attachment to the a, y, and 8 subunits (20).…”

Section: The Specificity Of [3h]tc Photoincorporation Into Thementioning

confidence: 99%

“…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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“…Points of attachment of ␣-neurotoxin within the nAChR binding sites have been examined by cross-linking chemically modified (8,9) or photoactivatable derivatives of ␣-neurotoxin (10 -13) and by simple ultraviolet irradiation without chemical modification (14). These labeling studies have suggested contacts with both ␣ and non-␣ subunits at the binding sites (see Refs.…”

mentioning

confidence: 99%

Subunit Interface Selectivity of the α-Neurotoxins for the Nicotinic Acetylcholine Receptor

Osaka

Malany

Kanter

et al. 1999

Journal of Biological Chemistry

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Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short ␣-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other ␣-neurotoxins, binds with high affinity to ␣␥ and ␣␦ subunit interfaces (K D ϳ100 pM) but binds with markedly reduced affinity to the ␣⑀ interface (K D ϳ100 nM). By constructing chimeras composed of portions of the ␥ and ⑀ subunits and coexpressing them with wild type ␣, ␤, and ␦ subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, ␥Pro-175 and ␥Glu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in ⑀, confer low affinity. To identify an interaction between ␥Glu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between ␥Glu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the ␥ subunit surface of the binding site interface. The nicotinic acetylcholine receptor (nAChR)1 found in muscle is a pentamer composed of four homologous subunits present in the stoichiometry ␣ 2 ␤␥␦ (fetal subtype) or ␣ 2 ␤⑀␦ (adult subtype). The subunits are arranged in a circular manner to surround a central channel in the order, ␣␥␣␦␤ or ␣⑀␣␦␤ (1-3). The two binding sites for agonists, competitive antagonists, and the slowly dissociating ␣-neurotoxins are formed at interfaces between the ␣␦ and ␣␥(⑀) subunit pairs. The extracellular domain in each subunit is formed principally from the aminoterminal 210 amino acids, which is followed by four membranespanning domains. Residues within the amino-terminal 210 amino acids have been shown to be the major contributors to the ligand binding sites and for dictating the order of assembly of subunits.Three segments of the ␣ subunit, well separated along the linear sequence, harbor major determinants for ligand binding; these segments contain the key residues around Tyr-93, between Trp-149 and Asp-152, and spanning the region from Val-188 through Asp-200 (see Refs. 3 and 4 for reviews). Similarly, four discontinuous segments of the non-␣ subunits, appearing on the opposite face of the subunit, contain major determinants for ligand selectivity; in the ␥ subunit these segments contain the key residues Lys-34, between Trp-55 and Gln-59, between Ser-111 and Tyr-117, and between Phe-172 and Asp-174.Since the early demonstration of irreversible neuromuscular blockade by the peptide from snake venom, ␣-bungarotoxin (5), and the use of labeled ␣-neurotoxins to identify the nAChR (6), these ...

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Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by .alpha.-neurotoxin crosslinking

Cited by 40 publications

References 37 publications

Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

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

Subunit Interface Selectivity of the α-Neurotoxins for the Nicotinic Acetylcholine Receptor

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