Functional Characterisation of Homomeric Ionotropic Glutamate Receptors GluR1-GluR6 in a Fluorescence-Based High Throughput Screening Assay

Abstract

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain

“…FabL9 was tested for modulation of GluA4 receptor by applying it in the presence of a saturating concentration of cyclothiazide to an HEK293 cell line stably expressing GluA4, and measuring Ca 2+ currents using a Fluo4-based fluorescence assay [35]. In this assay, FabL9 displayed neither agonist, antagonist, nor modulatory activity at the GluA4 receptor (data not shown).…”

Functional Characterization of Tet-AMPA [Tetrazolyl-2-amino-3-(3-hydroxy-5-methyl- 4-isoxazolyl)propionic Acid] Analogues at Ionotropic Glutamate Receptors GluR1−GluR4. The Molecular Basis for the Functional Selectivity Profile of 2-Bn-Tet-AMPA

“…The subcloning of rat GluR1 i and rat GluR4 i cDNAs from their original vectors into pcDNA3.1 has been described previously. 15 All mutant GluR1 i -pcDNA3.1 plasmids and the D399S/M686V/I687A-GluR1 i -pGEMHE-3Z plasmid were constructed using the QuikChange mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). The absence of unwanted mutations was verified by sequencing the cDNAs of all mutant GluRs.…”

“…The functional properties of the Tet-AMPA analogues were characterized at the stable GluR1 i -, GluR2Q i -, GluR3 i -, and GluR4 i -HEK293 cell lines and at the polyclonal HEK293 cells expressing WT and mutant GluRs in the Fluo-4/Ca 2+ assay as previously described. 15 The stable (monoclonal) and polyclonal HEK293 cells were split into poly-D-lysinecoated black 96-well plates with clear bottom (BD Biosciences, Bedford, MA). Later (16- The compounds were characterized in duplicate or triplicate (i.e., two or three data points for each concentration) at least three times (i.e., at least three different cell passages) at the WT or mutant GluRs.…”

High-Throughput Screen of GluK1 Receptor Identifies Selective Inhibitors with a Variety of Kinetic Profiles Using Fluorescence and Electrophysiology Assays

“…In addition to any compound having greater than 50% inhibition, any compound on the plate displaying greater % Inhibition than the 3-sigma cutoff was selected for further studies. Because this calcium flux protocol included a dye washout step, compounds were added via pintool prior to a FLIPR read, and it did not use a fluorescence quencher such as TR40, there 18,20 CNQX, 20 and UPB302 (Tocris Kd).…”