Chrisman Paul A, Podair Julie I, Jobe Emily M, Levandoski Mark M
Department of Chemistry, Programs in Biological Chemistry and Neuroscience, Grinnell College, 1116 8th Avenue, Grinnell, IA 50112, United States.
Department of Chemistry, Programs in Biological Chemistry and Neuroscience, Grinnell College, 1116 8th Avenue, Grinnell, IA 50112, United States.
Neuropharmacology. 2014 Apr;79:420-31. doi: 10.1016/j.neuropharm.2013.12.017. Epub 2013 Dec 25.
Allosteric modulation is a general feature of nicotinic acetylcholine receptors, yet the structural components and movements important for conversions among functional states are not well understood. In this study, we examine the communication between the binding sites for agonist and the modulator morantel (Mor) of neuronal α3β2 receptors, measuring evoked currents of receptors expressed in Xenopus oocytes with the two-electrode voltage-clamp method. We hypothesized that movement along an interface of β sheets connecting the agonist and modulator sites is necessary for allosteric modulation. To address this, we created pairs of substituted cysteines that span the cleft formed where the outer β sheet meets the β sheet constituting the (-)-face of the α3 subunit; the three pairs were L158C-A179C, L158C-G181C and L158C-K183C. Employing a disulfide trapping approach in which bonds are formed between neighboring cysteines under oxidation conditions, we found that oxidation treatments decreased the amplitude of currents evoked by either the agonist (ACh) or co-applied agonist and modulator (ACh + Mor), by as much as 51%, consistent with the introduced bond decreasing channel efficacy. Reduction treatment increased evoked currents up to 89%. The magnitude of the oxidation effects depended on whether agonists were present during oxidation and on the cysteine pair. Additionally, the cysteine mutations themselves decreased Mor potentiation, implicating these residues in modulation. Our findings suggest that these β sheets in the α3 subunit move with respect to each other during activation and modulation, and the residues studied highlight the contribution of this intramolecular allosteric pathway to receptor function.
变构调节是烟碱型乙酰胆碱受体的一个普遍特征，然而对于功能状态之间转换重要的结构成分和运动尚未得到很好的理解。在本研究中，我们研究了神经元α3β2受体激动剂结合位点与调节剂莫仑太尔（Mor）之间的通讯，采用双电极电压钳法测量非洲爪蟾卵母细胞中表达的受体诱发电流。我们假设沿着连接激动剂和调节剂位点的β折叠界面移动对于变构调节是必要的。为了解决这个问题，我们创建了跨越外β折叠与构成α3亚基（-）面的β折叠相遇处形成的裂隙的成对取代半胱氨酸；这三对分别是L158C-A179C、L158C-G181C和L158C-K183C。采用二硫键捕获方法，即在氧化条件下相邻半胱氨酸之间形成键，我们发现氧化处理使激动剂（ACh）或共同施加的激动剂和调节剂（ACh + Mor）诱发的电流幅度降低多达51%，这与引入的键降低通道效能一致。还原处理使诱发电流增加高达89%。氧化效应的大小取决于氧化过程中是否存在激动剂以及半胱氨酸对。此外，半胱氨酸突变本身降低了Mor的增强作用，表明这些残基参与调节。我们的研究结果表明，α3亚基中的这些β折叠在激活和调节过程中相互相对移动，并且所研究的残基突出了这种分子内变构途径对受体功能的贡献。
