Molecular Determinants of Ivermectin Sensitivity at the Glycine Receptor Chloride Channel
Molecular Determinants of Ivermectin Sensitivity at the Glycine Receptor Chloride Channel
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DOI：
10.1074/jbc.m111.262634
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发表时间：
2011-12-23
影响因子：
4.8
通讯作者：
Lynch, Joseph W.
中科院分区：
文献类型：
--
作者：
Lynagh, Timothy;Webb, Timothy I.;Lynch, Joseph W.
伊维菌素是一种驱虫药，通过激活线虫寄生虫中的谷氨酸门控氯离子通道受体（GluClRs）发挥作用。GluClR属于Cys环受体家族，其还包括甘氨酸受体（GlyR）氯离子通道。GluClRs和A288 G突变体GlyRs均被低纳摩尔浓度的伊维菌素激活。最近发表了秀丽隐杆线虫α GluClR与伊维菌素复合的晶体结构。在这里，我们探测伊维菌素敏感性决定因素的α 1 GlyR使用定点突变和电生理学。基于跨膜残基的诱变筛选，我们确定Ala（288）和Pro（230）为关键的敏感性决定因素。赛拉菌素和伊维菌素的作用的比较表明，高效力需要苯并呋喃C 05-OH。当与对接模拟一起考虑时，这些结果支持GlyR伊维菌素结合取向类似于GluClR晶体结构中所见。然而，尽管晶体结构显示伊维菌素通过与Leu（218）、Ser（260）和Thr（285）（α GluClR编号）的H-键与α GluClR相互作用，但我们的数据表明，与Ser（260）和Thr（285）同源的残基的H-键对于A288 G α 1 GlyR或三种其他GluClR中的伊维菌素高敏感性或直接激动剂功效并不重要。我们的数据还表明，伊维菌素二糖和GlyR M2-M3环残基之间的货车范德华相互作用对于伊维菌素的高敏感性是不重要的。因此，虽然我们的研究结果证实了伊维菌素的晶体结构所揭示的结合方向，他们表明，这种结构所揭示的一些结合相互作用不属于其他高度伊维菌素敏感的Cys环受体。
Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans alpha GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the alpha 1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala(288) and Pro(230) as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the alpha GluClR via H-bonds with Leu(218), Ser(260), and Thr(285) (alpha GluClR numbering), our data indicate that H-bonds with residues homologous to Ser(260) and Thr(285) are not important for high ivermectin sensitivity or direct agonist efficacy in A288G alpha 1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.