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 AcetylcholineS
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 m...
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Published in | Molecular pharmacology Vol. 76; no. 2; pp. 253 - 263 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
American Society for Pharmacology and Experimental Therapeutics
01.05.2009
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Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant DA017882]. Address correspondence to: Dr. Robert L. Rosenberg. University of North Carolina at Chapel Hill, Department of Pharmacology, CB 7365. Chapel Hill, NC 27599. E-mail: robert_rosenberg@med.unc.edu 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. |
ISSN: | 0026-895X 1521-0111 |
DOI: | 10.1124/mol.109.056226 |