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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Pharmacological Characterization of the Human Ionotropic Glutamate Receptor Subtype GluR3 Stably Expressed in Mammalian Cells

M. A. Varney, S. P. Rao, C. Jachec, C. Deal, S. D. Hess, L. P. Daggett, F.-F. Lin, E. C. Johnson and G. Veliçelebi
Journal of Pharmacology and Experimental Therapeutics April 1998, 285 (1) 358-370;
M. A. Varney
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S. P. Rao
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C. Jachec
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C. Deal
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S. D. Hess
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L. P. Daggett
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F.-F. Lin
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E. C. Johnson
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G. Veliçelebi
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Abstract

We have cloned the human ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR3 flip splice variant (hGluR3i) and developed a stable cell line expressing this receptor in HEK293 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 intracellular Ca++ ([Ca++]i) were observed in the presence of cyclothiazide, which attenuated receptor desensitization. [Ca++]i measurements were used to perform a detailed pharmacological characterization of hGluR3i with reference agonists and antagonists. The results of these studies showed that kainate and domoate were not fully efficacious agonists relative to glutamate. The binding affinities of agonists and competitive antagonists were determined in a [3H]AMPA competition binding assay. There was a good correlation between the functional data and the binding affinities obtained for competitive antagonists. However, the binding affinities of the agonists did not correlate with their functional EC50 values from [Ca++]i data, possibly because the binding assay predominantly measures the desensitized high-affinity state of the receptor. [3H]AMPA binding also was performed on membranes prepared from rat forebrain, and comparison of the data from HEK293 cells expressing hGluR3i and rat forebrain suggest that nearly all of the reference compounds show similar binding activities between the two membrane preparations, with the exception of fluoro-willardiine, kainate and 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2–3-dione (NBQX). These data suggest that cells stably expressing recombinant hGluR3irepresent pharmacologically valid experimental systems to study human AMPA receptors.

Footnotes

  • Send reprint requests to: Dr. Mark Varney, SIBIA Neurosciences, Inc., 505 Coast Blvd. South, La Jolla, CA 92037.

  • Abbreviations:
    AMOA
    (±)-2-amino-3-[3-(carboxymethoxy)-5-methyl-isoxazol-4-yl]propionic acid
    AMPA
    α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
    CNQX
    6-cyano-7-nitroquinoxaline-2,3-dione
    GAMS
    γ-d-glutamylaminomethylsulfonic acid
    DNQX
    6,7-dinitroquinoxaline-2,3-dione
    YM-90K
    6-(1H-imadazol-1-yl)-7-nitro-2,3-(1H, 4H)-quinoxalinedione
    GYKI-52466
    1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride
    HEPES
    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
    HBS
    HEPES-buffered saline
    hGluR3i
    human AMPA receptor subtype 3-flip
    NBQX
    6-nitro-7-sulfamoylbenzo(f)quinoxaline-2–3-dione
    NS 102
    6,7,8,9-tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime
    NS 257
    1,2,3,6,7,8-hexahydro-3-(hydroxyimino)-N,N,7-trimethyl-2-oxo-benzo[2,1-b:3,4-c′]dipyrrole-5-sulfonamide hydrochloride
    NMDA
    N-methyl-d-aspartate
    fluo-3/AM
    fluo-3-acetoxymethyl ester
    fura-2/AM
    fura-2-acetoxymethyl ester
    CTZ
    cyclothiazide
    nt
    nucleotide
    ANOVA
    analysis of variance
    • Received August 1, 1997.
    • Accepted December 29, 1997.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics
Vol. 285, Issue 1
1 Apr 1998
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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Pharmacological Characterization of the Human Ionotropic Glutamate Receptor Subtype GluR3 Stably Expressed in Mammalian Cells

M. A. Varney, S. P. Rao, C. Jachec, C. Deal, S. D. Hess, L. P. Daggett, F.-F. Lin, E. C. Johnson and G. Veliçelebi
Journal of Pharmacology and Experimental Therapeutics April 1, 1998, 285 (1) 358-370;

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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Pharmacological Characterization of the Human Ionotropic Glutamate Receptor Subtype GluR3 Stably Expressed in Mammalian Cells

M. A. Varney, S. P. Rao, C. Jachec, C. Deal, S. D. Hess, L. P. Daggett, F.-F. Lin, E. C. Johnson and G. Veliçelebi
Journal of Pharmacology and Experimental Therapeutics April 1, 1998, 285 (1) 358-370;
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