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A Novel Assay of G_i/o-Linked G Protein-Coupled Receptor Coupling to Potassium Channels Provides New Insights into the Pharmacology of the Group III Metabotropic Glutamate Receptors

Department of Pharmacology (C.M.N., K.A.M., Q.L., J.E.A., C.K., P.J.C., C.D.W.), Vanderbilt Institute of Chemical Biology (P.J.C., C.D.W.), Vanderbilt Program in Drug Discovery (C.M.N., P.J.C., C.D.W.); Vanderbilt Molecular Libraries Screening Center Networks Center for GPCRS, Ion Channels, and Transporters (P.J.C., C.D.W.), Vanderbilt University, Nashville, Tennessee

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 Gβ 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.

Address correspondence to: Colleen M. Niswender, 1215 MRB IV, Department of Pharmacology, Vanderbilt University, Nashville, TN 37232. E-mail: colleen.niswender{at}vanderbilt.edu

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