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Molecular Pharmacology

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Research ArticleArticle

Identification of Benzodiazepine Binding Site Residues in the γ2 Subunit of the γ-Aminobutyric AcidA Receptor

Amy M. Kucken, David A. Wagner, Peter R. Ward, Jeremy A. Teissére, Andrew J. Boileau and Cynthia Czajkowski
Molecular Pharmacology May 2000, 57 (5) 932-939;
Amy M. Kucken
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David A. Wagner
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Peter R. Ward
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Jeremy A. Teissére
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Andrew J. Boileau
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Cynthia Czajkowski
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Abstract

γ-Aminobutyric acidA receptor γ-subunits are important for benzodiazepine (BZD) binding and modulation of the γ-aminobutyric acid-mediated Cl− current. Previously, by using γ2/α1 chimeric subunits, we identified two domains of the γ2-subunit, Lys-41-Trp-82 and Arg-114-Asp-161, that are, in conjunction, necessary and sufficient for high-affinity BZD binding. In this study, we generated additional γ2/α1 chimeric subunits and γ2 point mutants to identify specific residues within the γ2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant γ2 and γ2/α1 chimeric subunits were expressed with wild-type α1 and β2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the γ2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, γ2 Met-57 and γ2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, γ2 Ala-79, that not only is important for high-affinity binding by flunitrazepam (a strong positive modulator) but also plays a crucial role in the binding of the imidazobenzodiazepines Ro15-1788 (a zero modulator) and Ro15-4513 (a weak negative modulator) in the BZD binding pocket. Results from site-directed mutagenesis of γ2 Ala-79 suggest that this residue may be part of a microdomain within the BZD binding site that is important for binding imidazobenzodiazepines. This separation of drug-specific microdomains for competitive BZD ligands lends insight into the structural determinants governing the divergent effects of these compounds.

Footnotes

  • Send reprint requests to: Cynthia Czajkowski, Ph.D., Department of Physiology, University of Wisconsin-Madison, 1300 University Ave., MSC Room 197A, Madison, WI 53706. E-mail:czajkowski{at}physiology.wisc.edu

  • This work was supported in part by grants to C.C. from the Alcoholic Beverage Association and NINDS-the National Institutes of Health. C.C. is a recipient of the Burroughs Wellcome Fund New Investigator Award in the Basic Pharmacological Sciences.

  • Abbreviations:
    GABA
    γ-aminobutyric acid
    PCR
    polymerase chain reaction
    BZD
    benzodiazepine
    • Received October 14, 1999.
    • Accepted January 5, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 57 (5)
Molecular Pharmacology
Vol. 57, Issue 5
1 May 2000
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Research ArticleArticle

Identification of Benzodiazepine Binding Site Residues in the γ2 Subunit of the γ-Aminobutyric AcidA Receptor

Amy M. Kucken, David A. Wagner, Peter R. Ward, Jeremy A. Teissére, Andrew J. Boileau and Cynthia Czajkowski
Molecular Pharmacology May 1, 2000, 57 (5) 932-939;

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Research ArticleArticle

Identification of Benzodiazepine Binding Site Residues in the γ2 Subunit of the γ-Aminobutyric AcidA Receptor

Amy M. Kucken, David A. Wagner, Peter R. Ward, Jeremy A. Teissére, Andrew J. Boileau and Cynthia Czajkowski
Molecular Pharmacology May 1, 2000, 57 (5) 932-939;
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