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

Abstract

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␤␥ 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 GIRKmediated 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.The metabotropic glutamate receptors (mGluRs) are among the most abundantly expressed receptors in the mammalian central nervous system and are thought to play critical roles in regulating activity in a broad range of CNS circuits (Conn and Pin, 1997). Despite an increasing appreciation of the involvement of mGluRs in CNS function, however, progress in understanding the signaling pathways and functional properties of some members of the mGluR family has been relatively slow. This is especially true for the group III mGluRs, which include mGluRs 4, 6, 7, and 8.

ML297 (VU0456810), the First Potent and Selective Activator of the GIRK Potassium Channel, Displays Antiepileptic Properties in Mice

“…HEK-293 cells coexpressing human GIRK1/2 and rat mGlu 8 were generated as previously described. 25 A HEK-293 cell line (ATCC, Manassas, VA) expressing human GIRK1 and human GIRK2 was constructed by transfecting HEK-293 cells with GIRK1 and GIRK2 (Origene, Rockville, MD) cloned into the pBudCE4.1 vector (Life Technologies, Carlsbad, CA) using FuGene 6 (Promega, Madison, WI) transfection reagent. Unless otherwise noted, other ion channel-expressing cells were prepared by transfecting HEK-293 cells with individual or combinations of subunits: GIRK1, pCMV6-A-BSD; GIRK2, pCMV6-A-puro; GIRK3, pCMV6-A-hygro; GIRK 4, pCMV6-A-AC; Kv7.4, pIRESneo3.…”

Direct control of Na+-K+-2Cl−-cotransport protein (NKCC1) expression with aldosterone

“…We utilized this methodology following the protocol of the Delpire group (21), since they and other groups have shown similar results between 86 Rb and FluxOR. This method uses TI ϩ as a surrogate of K ϩ , and a TI ϩ -sensitive fluorescent dye (Flix-ORTM) to visualize TI ϩ uptake through NKCC1 in single cells (27,38,59). We found that addition of ALD to the media resulted in a marked increase in fluorescence of NKCC1 with both the 86 Rb and Tl ϩ methodologies.…”

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

“…11,27 Nevertheless, the potential of this assay to quickly test multiple agonists or putative modulators of receptor desensitization in an intact system makes it an attractive option. GIRK channel activation has most commonly been assessed using electrophysiological techniques, but assays potentially suitable for HTS have been reported using thallium flux 29 or commercially available membrane potential dyes. 30,31 Thallium is toxic and apparently unsuitable for all cell lines, 31 whereas other membrane potential sensitive dyes (Di-Bac, HLB 021-152) give results qualitatively similar to those reported here.…”