Roles of Agonist-Binding Sites in Nicotinic Acetylcholine Receptor Function

Dunn, Susan M. J.; Raftery, Michael A.

doi:10.1006/bbrc.2000.3960

Cited by 9 publications

(5 citation statements)

References 33 publications

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“…We identified low affinity agonist-specific sites, which displayed properties that were consistent with the involvement of these sites in channel activation [12][13][14]. Supporting evidence for multiple binding sites has come from studies of α-dendrotoxin binding [15], photoaffinity labeling [16] and from flux studies of pre-formed receptor-ligand complexes [17]. In this report, we have characterized the binding of an agonist, epibatidine [18], to the Torpedo nAChR and directly demonstrate the presence of four agonist binding sites under equilibrium conditions.…”

Section: Introductionmentioning

confidence: 67%

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

Kawai

Dunn

Raftery

2008

Biochemical and Biophysical Research Communications

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The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabelled ligand binding techniques, we show that the binding of

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Section: Introductionmentioning

confidence: 67%

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

Kawai

Dunn

Raftery

2008

Biochemical and Biophysical Research Communications

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“…It remains to be established whether this reflects the presence of distinct low affinity sites that mediate channel opening as we have proposed for the Torpedo nAChR (see Ref. 28). …”

Section: Figmentioning

confidence: 85%

“…rapid agonist-induced flux measurements using native Torpedo membrane vesicles (28). Under equilibrium conditions, it is well established that this receptor carries two high affinity sites for agonists (K D of ϳ100 nM for carbamylcholine), and it is generally assumed that occupancy of these sites in the resting state of the receptor leads to the equilibrium desensitized state (29).…”

Section: Figmentioning

confidence: 99%

Functional Consequences of the Loss of High Affinity Agonist Binding to γ-Aminobutyric Acid Type A Receptors

Newell¹,

Dunn²

2002

Journal of Biological Chemistry

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We reported previously that tyrosine 62 of the ␤2 subunit of the ␥-aminobutyric acid, type A (GABA A ) receptor is an important determinant of high affinity agonist binding and that recombinant ␣1␤2␥2 L receptors carrying the Y62S mutation lack measurable high affinity sites for [ 3 H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC 50 ϳ0.7 M, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the ␤2 Y62S and Y62F mutations desensitized at slightly higher (2-7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABA A receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed.

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“…In previous studies of the native Torpedo nAChR, we made use of covalently bound fluorescent probes to monitor agonist binding. One probe, IANBD (4-{[(iodoacetoxy)ethyl]methylamino}-7-nitro-2,1,3-benzoxadiazole) ( − ), appears to monitor binding to low-affinity sites that we have correlated with channel activation ( , ). Curiously, unlike all other agonists that have been examined, the binding of SbCh to fluorescently labeled nAChR was biphasic, suggesting the presence of two classes of binding sites with dissociation constants of ∼2 μM and ∼2 mM ().…”

Section: Discussionmentioning

confidence: 99%

Tryptophan 86 of the α Subunit in the Torpedo Nicotinic Acetylcholine Receptor Is Important for Channel Activation by the Bisquaternary Ligand Suberyldicholine

Kapur¹,

Davies²,

Dryden³

et al. 2006

Biochemistry

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Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

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Roles of Agonist-Binding Sites in Nicotinic Acetylcholine Receptor Function

Cited by 9 publications

References 33 publications

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

Functional Consequences of the Loss of High Affinity Agonist Binding to γ-Aminobutyric Acid Type A Receptors

Tryptophan 86 of the α Subunit in the Torpedo Nicotinic Acetylcholine Receptor Is Important for Channel Activation by the Bisquaternary Ligand Suberyldicholine

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