PHARMACOLOGICAL CHARACTERIZATION OF THE HUMAN IONOTROPIC GLUTAMATE-RECEPTOR SUBTYPE GLUR3 STABLY EXPRESSED IN MAMMALIAN-CELLS

We have cloned the human ionotropic lpha-amino-3-hydroxy-5-methyl-4-is oxazolepropionic acid (AMPA) receptor GluR3 flip splice variant (hGluR 3(i)) and developed a stable cell line expressing this receptor in HEK 293 cells. Electrophysiological recordings demonstrated that glutamate -evoked currents desensitize rapidly, with a mean desensitization time constant of 5.4 ms. Robust glutamate-evoked increases in intracellula r Ca++ ([Ca++](i)) were observed in the presence of cyclothiazide, whi ch attenuated receptor desensitization. [Ca++](i) measurements were us ed to perform a detailed pharmacological characterization of hGluR3(i) with reference agonists and antagonists. The results of these studies showed that kainate and domoate were not fully efficacious agonists r elative to glutamate. The binding affinities of agonists and competiti ve antagonists were determined in a [H-3]AMPA competition binding assa y. There was a good correlation between the functional data and the bi nding affinities obtained for competitive antagonists. However, the bi nding affinities of the agonists did not correlate with their function al EC50 values from [Ca++], data, possibly because the binding assay p redominantly measures the desensitized high-affinity state of the rece ptor. [H-3]AMPA binding also was performed on membranes prepared from rat forebrain, and comparison of the data from HEK293 cells expressing hGluR3(i) and rat forebrain suggest that nearly all of the reference compounds show similar binding activities between the two membrane pre parations, with the exception of fluoro-willardiine, kainate and 6-nit ro-7-sulfamoylbenzo(f)quinoxaline-2-3-dione (NBQX). These data suggest that cells stably expressing recombinant hGluR3(i) represent pharmaco logically valid experimental systems to study human AMPA receptors.