Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay.
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.
Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Mette
M
Strange
Hans
H
Bräuner-Osborne
Anders A
AA
Jensen
D000814:Aniline Compounds / Q000032:analysis / Q000378:metabolism; D001581:Benzothiadiazines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D020650:Combinatorial Chemistry Techniques; D003208:Concanavalin A / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004594:Electrophysiology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005453:Fluorescence; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D018091:Receptors, AMPA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017470:Receptors, Glutamate / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014966:Xanthenes / Q000032:analysis / Q000378:metabolism
D000814:Aniline Compounds; D001581:Benzothiadiazines; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; C409648:Fluo 4; D011810:Quinoxalines; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D014966:Xanthenes; D003208:Concanavalin A; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; D018698:Glutamic Acid; C004639:cyclothiazide; D007608:Kainic Acid; D002118:Calcium