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
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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
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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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