A novel assay of Gi/o-Linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors
A novel assay of Gi/o-Linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors
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DOI：
10.1124/mol.107.041053
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发表时间：
2008-04-01
影响因子：
3.6
通讯作者：
Weaver, C. David
中科院分区：
文献类型：
--
作者：
Niswender, Colleen M.;Johnson, Kari A.;Weaver, C. David
第三类代谢性谷氨酸受体(MGluRs)代表了一类突触前表达的G蛋白偶联受体(GPCRs)，具有巨大的治疗潜力；然而，很难建立强有力的细胞分析来研究它们的功能。我们提出了一种与传统的高通量筛选平台兼容的新方法，用于检测与G(I/O)偶联GPCRs相互作用的药理配体的活性，包括第III族mGluRs4、7和8。该方法利用G(I)和G(O)异三聚体的Gβ伽马亚基与G蛋白调节的内向整流钾通道(GIRKs)相互作用的能力，我们在这里展示了我们能够检测多种类型的药效团的活性，包括几种不同GPCRs的激动剂、拮抗剂和变构调制剂。使用GIRK介导的铊通量，我们对一些商业上可用的化合物的活性进行了并排比较，其中一些还没有得到广泛的评估，因为以前在三个主要的III族mGluR中的每一个都缺乏强有力的分析。值得注意的是，几个以前被认为是通用的III类mGluR拮抗剂的化合物在本实验中的活性非常弱，这表明这些化合物可能在天然系统中不能有效地抑制这些受体。我们预计，GIRK介导的Tl通量策略将为推进G(i/o)偶联GPCR生物学的研究和促进配体的发现和表征提供一种新的工具。
The group III metabotropic glutamate receptors (mGluRs) represent a family of presynaptically expressed G-protein-coupled receptors (GPCRs) with enormous therapeutic potential; however, robust cellular assays to study their function have been difficult to develop. We present here a new assay, compatible with traditional high-throughput screening platforms, to detect activity of pharmacological ligands interacting with G(i/o)-coupled GPCRs, including the group III mGluRs 4, 7, and 8. The assay takes advantage of the ability of the G beta gamma subunits of G(i) and G(o) heterotrimers to interact with G-protein regulated inwardly rectifying potassium channels (GIRKs), and we show here that we are able to detect the activity of multiple types of pharmacophores including agonists, antagonists, and allosteric modulators of several distinct GPCRs. Using GIRK-mediated thallium flux, we perform a side-by-side comparison of the activity of a number of commercially available compounds, some of which have not been extensively evaluated because of the previous lack of robust assays at each of the three major group III mGluRs. It is noteworthy that several compounds previously considered to be general group III mGluR antagonists have very weak activity using this assay, suggesting the possibility that these compounds may not effectively inhibit these receptors in native systems. We anticipate that the GIRK-mediated thallium flux strategy will provide a novel tool to advance the study of G(i/o)-coupled GPCR biology and promote ligand discovery and characterization.