Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

TY - JOUR

T1 - Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

AU - Strange, Mette

AU - Bräuner-Osborne, Hans

AU - Jensen, Anders A.

PY - 2006

Y1 - 2006

N2 - 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.

AB - 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.

KW - Aniline Compounds

KW - Benzothiadiazines

KW - Calcium

KW - Cell Line

KW - Combinatorial Chemistry Techniques

KW - Concanavalin A

KW - Drug Evaluation, Preclinical

KW - Electrophysiology

KW - Excitatory Amino Acid Agonists

KW - Excitatory Amino Acid Antagonists

KW - Fluorescence

KW - Glutamic Acid

KW - Humans

KW - Kainic Acid

KW - Quinoxalines

KW - Receptors, AMPA

KW - Receptors, Glutamate

KW - Signal Transduction

KW - Xanthenes

M3 - Journal article

C2 - 16475972

SN - 1386-2073

VL - 9

SP - 147

EP - 158

JO - Combinatorial Chemistry and High Throughput Screening

JF - Combinatorial Chemistry and High Throughput Screening

IS - 2

ER -