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
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
复制标题
DOI：
10.1124/mol.109.056226
复制
发表时间：
2009-08-01
影响因子：
3.6
通讯作者：
Rosenberg, Robert L.
中科院分区：
文献类型：
--
作者：
Barron, Sean C.;McLaughlin, James T.;Rosenberg, Robert L.
尼古丁乙酰胆碱受体与几种神经精神疾病有关，包括尼古丁成瘾、阿尔茨海默病、精神分裂症和抑郁症。因此，它们代表了药物开发和靶向治疗干预的关键分子靶标。了解变构调节剂增强这些受体活化的分子机制对新药的开发至关重要。我们使用取代的半胱氨酸可及性方法研究了正变构调节剂N-（5-氯-2，4-二甲氧基苯基）-N '-（5-甲基-3-异恶唑基）脲（PNU-120596）在携带L247 T突变的α 7烟碱受体的细胞外配体结合结构域中诱导的构象变化。PNU-120596导致内β折叠、过渡区和激动剂结合位点的半胱氨酸可及性发生变化。这些可及性的变化与单独由ACh引起的变化相似但不完全相同。特别是，PNU-120596在N170 C（过渡区）诱导MTSEA可及性的变化，这与乙酰胆碱（ACh）诱发的变化基本不同。我们发现，PNU-120596在不存在变构调节的情况下诱导位置E172 C的变化。我们鉴定了激动剂结合位点（W148 C）的半胱氨酸突变，该突变表现出意想不到的表型，其中PNU-120596作为完全激动剂。在该突变体中，乙酰胆碱诱发的电流比PNU诱发的电流对巯基修饰更敏感，表明PNU-120596在未被占据的激动剂结合位点不结合。我们的研究结果提供的证据表明，PNU-120596的结合位点不在激动剂结合位点，并表明，积极的变构调节剂，如PNU-120596增强激动剂诱发的门控烟碱受体引起的构象效应是相似的，但不相同的门控构象促进乙酰胆碱。
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 alpha 7 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.