Differential Agonist-Induced Displacement of Quinacrine and Ethidium from Their Respective Histrionicotoxin-Sensitive Binding Sites on the Torpedo Acetylcholine Receptor

Hugo R. Arias, David A. Johnson

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Fluorescence spectroscopy was used to begin to localize the agonist inhibitory binding site on the nicotinic acetylcholine receptor (AcChR) from Torpedo califomica. High concentrations of three cholinergic agonists, suberyldicholine (SubCh), acetylcholine (AcCh), and carbamylcholine (CCh), differentially inhibited the binding of two noncompetitive inhibitors (NCIs), quinacrine and ethidium, which bind at distinctly different loci on the desensitized AcChR at zero membrane potential. The agonistinduced inhibition of quinacrine binding occurred at significantly lower (17-fold) concentrations than the inhibition of ethidium binding. Schild plots of SubCh inhibition of ethidium and quinacrine binding showed the competitive nature of the agonist inhibition of the binding of these two NCIs. The quenching constants for short-range quenching of receptor-bound quinacrine and ethidium fluorescence by spinlabeled acetylcholine were about the same as their inhibition constants for agonist-induced displacement of AcChR-bound quinacrine and ethidium. The results demonstrate that agonists can directly bind to both the quinacrine and the ethidium binding sites, albeit at different agonist concentrations. Because the agonist-induced displacement of receptor-bound quinacrine occurs at significantly lower concentrations than the displacement of ethidium, the quinacrine binding site is more likely than the ethidium binding site to form part of the agonist inhibitory binding site.

Original languageEnglish
Pages (from-to)1589-1595
Number of pages7
JournalBiochemistry
Volume34
Issue number5
DOIs
StatePublished - 1 Jan 1995
Externally publishedYes

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Torpedo
Quinacrine
Ethidium
Cholinergic Receptors
Binding Sites
Acetylcholine
Quenching
Cholinergic Agonists
Competitive Binding
histrionicotoxin
Fluorescence Spectrometry
Fluorescence spectroscopy
Nicotinic Receptors
Carbachol
Membrane Potentials
Fluorescence
Membranes

Cite this

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title = "Differential Agonist-Induced Displacement of Quinacrine and Ethidium from Their Respective Histrionicotoxin-Sensitive Binding Sites on the Torpedo Acetylcholine Receptor",
abstract = "Fluorescence spectroscopy was used to begin to localize the agonist inhibitory binding site on the nicotinic acetylcholine receptor (AcChR) from Torpedo califomica. High concentrations of three cholinergic agonists, suberyldicholine (SubCh), acetylcholine (AcCh), and carbamylcholine (CCh), differentially inhibited the binding of two noncompetitive inhibitors (NCIs), quinacrine and ethidium, which bind at distinctly different loci on the desensitized AcChR at zero membrane potential. The agonistinduced inhibition of quinacrine binding occurred at significantly lower (17-fold) concentrations than the inhibition of ethidium binding. Schild plots of SubCh inhibition of ethidium and quinacrine binding showed the competitive nature of the agonist inhibition of the binding of these two NCIs. The quenching constants for short-range quenching of receptor-bound quinacrine and ethidium fluorescence by spinlabeled acetylcholine were about the same as their inhibition constants for agonist-induced displacement of AcChR-bound quinacrine and ethidium. The results demonstrate that agonists can directly bind to both the quinacrine and the ethidium binding sites, albeit at different agonist concentrations. Because the agonist-induced displacement of receptor-bound quinacrine occurs at significantly lower concentrations than the displacement of ethidium, the quinacrine binding site is more likely than the ethidium binding site to form part of the agonist inhibitory binding site.",
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AB - Fluorescence spectroscopy was used to begin to localize the agonist inhibitory binding site on the nicotinic acetylcholine receptor (AcChR) from Torpedo califomica. High concentrations of three cholinergic agonists, suberyldicholine (SubCh), acetylcholine (AcCh), and carbamylcholine (CCh), differentially inhibited the binding of two noncompetitive inhibitors (NCIs), quinacrine and ethidium, which bind at distinctly different loci on the desensitized AcChR at zero membrane potential. The agonistinduced inhibition of quinacrine binding occurred at significantly lower (17-fold) concentrations than the inhibition of ethidium binding. Schild plots of SubCh inhibition of ethidium and quinacrine binding showed the competitive nature of the agonist inhibition of the binding of these two NCIs. The quenching constants for short-range quenching of receptor-bound quinacrine and ethidium fluorescence by spinlabeled acetylcholine were about the same as their inhibition constants for agonist-induced displacement of AcChR-bound quinacrine and ethidium. The results demonstrate that agonists can directly bind to both the quinacrine and the ethidium binding sites, albeit at different agonist concentrations. Because the agonist-induced displacement of receptor-bound quinacrine occurs at significantly lower concentrations than the displacement of ethidium, the quinacrine binding site is more likely than the ethidium binding site to form part of the agonist inhibitory binding site.

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