The Nicotinic Acetylcholine Receptor as a Model for a Superfamily of Ligand-Gated Ion Channel Proteins

McLane, Kathryn E.; Dunn, Shelagh; Manfredi, A.A.; Conti‐Tronconi, Bianca M.; Raftery, Michael A.

doi:10.1016/b978-012159640-8/50011-5

Cited by 5 publications

(6 citation statements)

References 312 publications

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“…From analysis of the multiple conformational transitions observed, we proposed a model involving multiple ligand binding steps (Scheme 1) in which, under some conditions i.e., high agonist concentration, more than two binding sites per receptor molecule could be occupied. However in numerous equi-librium ligand binding studies [reviewed in Stroud et al (1990), Changeux et al (1992), andMcLane et al (1996)], the stoichiometry of agonist binding sites has been demonstrated to be two sites per AcChR molecule. In an attempt to resolve this discrepancy, we have studied the dissociation kinetics of the radiolabeled agonists, [ 3 H]AcCh and [ 3 H]-SdCh, using rapid filtration methods following extensive dilution of the receptor-agonist complex.…”

Section: Discussionmentioning

confidence: 99%

“…A prediction of this mechanism is therefore that more than two binding sites will be occupied in the fully desensitized receptor at high agonist concentrations, i.e., in complex C 2 . However, in direct binding studies carried out in many different laboratories, the stoichiometry of high-affinity binding sites for [ 3 H]AcCh or [ 3 H]Carb has been found to be two per nAcChR [reviewed by Stroud et al (1990), Changeux et al (1992), andMcLane et al (1996)].…”

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confidence: 99%

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Agonist Binding to the Torpedo Acetylcholine Receptor. 1. Complexities Revealed by Dissociation Kinetics

Dunn

Raftery

1997

Biochemistry

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Examination of the kinetics of dissociation of [3H]acetylcholine and [3H]suberyldicholine from the membrane-bound acetylcholine receptor from Torpedo californica has revealed complexities in the high-affinity binding of nicotinic agonists. Each agonist binds to two high-affinity sites per receptor with an equilibrium dissociation constant of approximately 15 nM. When dissociation of [3H]acetylcholine from the receptor complex was triggered by dilution, dissociation occurred as a monophasic process with an apparent rate of 0.023 +/- 0.010 s(-1). However, when micromolar concentrations of unlabeled agonists (acetylcholine, carbamylcholine or suberyldicholine) were included in the dilution buffer this rate increased about 5-fold. This accelerating effect occurred even when the two high-affinity sites were initially saturated with the radioligand. This suggested the presence of an additional site (or subsite) for agonist with affinity in the micromolar range. However, at concentrations of 0-20 microM, no additional sites for [3H]acetylcholine were detected at equilibrium. To explain these results, we propose that each high-affinity site is made up of two subsites, A and B, which are mutually exclusive at equilibrium. With [3H]acetylcholine initially occupying site A, occupancy of site B by unlabeled ligand reduces the affinity for site A and accelerates the dissociation of the radioligand. Studies of dissociation of [3H]suberyldicholine, a large bis-quaternary agonist, provide some clue as to the possible physical nature of these subsites. Whereas its dissociation rate was similar to that of [3H]acetylcholine (0.028 +/- 0.012 s(-1)), this rate was only marginally, if at all, affected by the presence of unlabeled ligands. These results, in addition to those presented in the accompanying manuscript, lead to the proposal that [3H]suberyldicholine is able to cross-link the two subsites or at least sterically occlude the second site.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Agonist Binding to the Torpedo Acetylcholine Receptor. 1. Complexities Revealed by Dissociation Kinetics

Dunn

Raftery

1997

Biochemistry

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“…Clearly the loop C that contains highly functional residues conserved in both receptors plays a predominant binding role. The evidence that supports that conclusion includes (i) mutational analyses with short chain (33,34) and long chain (35)(36)(37) toxins, (ii) the use of synthetic receptor peptides of the region 180-200 (38,39), (iii) studies on the resistance of various species to toxins (40,41), (iv) direct affinity labeling experiments (42), and (v) resolution of solution structures of the complexes formed between ␣-Bgtx and peptides (32,(43)(44)(45). Also, the additional binding function observed here for other ␣ and non-␣ loops was postulated for muscular receptors (46)(47)(48).…”

Section: Discussionmentioning

confidence: 99%

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

Fruchart-Gaillard

Gilquin

Antil-Delbeke

et al. 2002

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

120

150

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To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the ␣-cobratoxin-␣7 receptor complex. This model was achieved by using (i) a three-dimensional model of the ␣7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the ␣-cobratoxin and ␣7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. T he ␣-neurotoxins from snake venom are potent antagonists that block nicotinic acetylcholine receptors (AChRs) and hence affect synaptic transmission (1-3). Despite many studies (reviewed in ref. 4), the molecular process associated with this efficient blockage remains unclear. To approach this question, we previously studied ␣-cobratoxin (␣-Cbtx), an ␣͞K neurotoxin that binds to both muscular and homopentameric neuronal receptors (␣7 and ␣8) with high affinities (4). This toxin, similar to other snake neurotoxins, is folded into three adjacent loops rich in ␤-sheet that emerge from a small globular core in which four disulfide bonds are located (5). By mutational analyses, the residues by which ␣-Cbtx interacts with the muscular-type or neuronal ␣7 receptors were identified previously (6, 7). The present study shows how functional residues account for the antagonistic properties of the toxin toward the ␣7 neuronal receptor. The ␣7 AChR possesses five identical ␣7 subunits (8) that offer five ligand-binding sites located at the interface of two subunits (9). These sites include residues located on the different functional loops described previously on the principal ␣7 (ϩ) face, loops A, B, and C and on the complementary ␣7 (Ϫ) face, loops D, E, and F (refs. 10-13; see Fig. 1). Until now, the residues of the ␣7 receptor involved in snake toxin binding have remained unknown.The aim of the present paper is fourfold. First, by an extensive mutational study we have identified ␣7 receptor residues involved in the interaction with ␣-Cbtx. Second, by using a double-mutant cycle approach we have disclosed several pairs of interacting residues in the toxin-receptor complex. Third, by using the three-dimensional (3D) structure of an AChBP that is similar functionally and structurally to the N-terminal domain of an AChR ␣-subunit (14), we used a 3D model for the ␣7 subunit extracellular region obtained by comparative modeling [see accompanying paper on page 3210 (15)]. Fourth, by using this model, a molecular dynamics simulation of the loop F region, and the constraints derived from our pairwise analysis, we propose an experimentally based 3D model of the complex between the ␣-Cbtx and ␣7 receptor, which explains the antagonistic properties of the snake toxin toward the neuronal recepto...

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“…The Torpedo nicotinic acetylcholine receptor (nAcChR) 1 is a ligand-gated ion channel composed of four subunits with a stoichiometry of R 2 βγδ (reviewed in 1) and resembles most closely the muscle-type nAcChR. Two agonists bind to activation sites and open a channel and induce cation flux, which results in depolarization of postsynaptic cell membranes.…”

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confidence: 99%

“…Two agonists bind to activation sites and open a channel and induce cation flux, which results in depolarization of postsynaptic cell membranes. Abundance of this receptor in the electric tissue has allowed extensive biochemical studies and this nAcChR serves as a model for other ligand-gated ion channels, which include neuronal nAcChR, γ-aminobutyric acid type A (GABA A ), glycine, and 5-hydroxytryptamine type 3 (5-HT 3 ) receptors ( , ).…”

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confidence: 99%

Eserine and Other Tertiary Amine Interactions with Torpedo Acetylcholine Receptor Postsynaptic Membrane Vesicles

Kawai

Carlson²,

Okita³

et al. 1998

Biochemistry

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Interaction of the tertiary amines, arecolone, eserine (physostigmine), (+)-epibatidine, and (+/-)-epibatidine, with Torpedo nicotinic acetylcholine receptor-enriched membrane vesicles was investigated to characterize their action on the receptor, using stopped-flow thallium (I)-flux spectrofluorimetry. Arecolone, (+)-epibatidine, and (+/-)-epibatidine were agonists with activation constants of 390, 19, and 39 microM, respectively. Eserine was not an agonist but rather an antagonist for agonist-induced activation of the receptor with an inhibition constant of approximately 150 microM. The choice of the fluorescent dye used (entrapped within the membrane vesicles) was critical for interpretation of the effects of eserine. With 1,3,6,8-pyrene tetrasulfate (PTS), eserine appeared to act as an agonist. However, it was shown that such an effect was caused by rapid diffusion of the uncharged form of the amine across the membrane followed by direct interaction with PTS rather than eserine-induced cation transport. The use of a different fluorescent dye, 8-aminonaphthaline-1,3,6-trisulfate, with which eserine does not interact allowed demonstration of the action of eserine as an antagonist rather than as an agonist.

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The Nicotinic Acetylcholine Receptor as a Model for a Superfamily of Ligand-Gated Ion Channel Proteins

Cited by 5 publications

References 312 publications

Agonist Binding to the Torpedo Acetylcholine Receptor. 1. Complexities Revealed by Dissociation Kinetics

Agonist Binding to the Torpedo Acetylcholine Receptor. 1. Complexities Revealed by Dissociation Kinetics

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

Eserine and Other Tertiary Amine Interactions with Torpedo Acetylcholine Receptor Postsynaptic Membrane Vesicles

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