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Molecular Pharmacology

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Research ArticleArticle

An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine

Sean C. Barron, James T. McLaughlin, Jennifer A. See, Vanessa L. Richards and Robert L. Rosenberg
Molecular Pharmacology August 2009, 76 (2) 253-263; DOI: https://doi.org/10.1124/mol.109.056226
Sean C. Barron
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James T. McLaughlin
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Jennifer A. See
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Vanessa L. Richards
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Robert L. Rosenberg
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Abstract

Nicotinic acetylcholine receptors are implicated in several neuropsychiatric disorders, including nicotine addiction, Alzheimer's, schizophrenia, and depression. Therefore, they represent a critical molecular target for drug development and targeted therapeutic intervention. Understanding the molecular mechanisms by which allosteric modulators enhance activation of these receptors is crucial to the development of new drugs. We used the substituted cysteine accessibility method to study conformational changes induced by the positive allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596) in the extracellular ligand binding domain of α7 nicotinic receptors carrying the L247T mutation. PNU-120596 caused changes in cysteine accessibility at the inner beta sheet, transition zone, and agonist binding site. These changes in accessibility are similar to but not identical to those caused by ACh alone. In particular, PNU-120596 induced changes in MTSEA accessibility at N170C (in the transition zone) that were substantially different from those evoked by acetylcholine (ACh). We found that PNU-120596 induced changes at position E172C in the absence of allosteric modulation. We identified a cysteine mutation of the agonist binding site (W148C) that exhibited an unexpected phenotype in which PNU-120596 acts as a full agonist. In this mutant, ACh-evoked currents were more sensitive to thiol modification than PNU-evoked currents, suggesting that PNU-120596 does not bind at unoccupied agonist-binding sites. Our results provide evidence that binding sites for PNU-120596 are not in the agonist-binding sites and demonstrate that positive allosteric modulators such as PNU-120596 enhance agonist-evoked gating of nicotinic receptors by eliciting conformational effects that are similar but nonidentical to the gating conformations promoted by ACh.

Footnotes

  • This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant DA017882].

  • ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; TMD, transmembrane domain; PAM, positive allosteric modulator; PNU-120596, N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea; LBD, ligand binding domain; SCAM, substituted cysteine accessibility method; ESLC, extracellular solution, low calcium; MTS, methanethiosulfonate; MTSEA, 2-aminoethylmethanethiosulfonate; ACh, acetylcholine; C-O, closed-open gating transition; WT, wild type.

  • ↵ Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

    • Accepted May 1, 2009.
    • Received March 13, 2009.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 76 (2)
Molecular Pharmacology
Vol. 76, Issue 2
1 Aug 2009
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An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine
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Research ArticleArticle

An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine

Sean C. Barron, James T. McLaughlin, Jennifer A. See, Vanessa L. Richards and Robert L. Rosenberg
Molecular Pharmacology August 1, 2009, 76 (2) 253-263; DOI: https://doi.org/10.1124/mol.109.056226

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Research ArticleArticle

An Allosteric Modulator of α7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine

Sean C. Barron, James T. McLaughlin, Jennifer A. See, Vanessa L. Richards and Robert L. Rosenberg
Molecular Pharmacology August 1, 2009, 76 (2) 253-263; DOI: https://doi.org/10.1124/mol.109.056226
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