Molecular Dissection of Benzodiazepine Binding and Allosteric Coupling Using Chimeric γ-Aminobutyric AcidA Receptor Subunits
Abstract
Although γ-aminobutyric acid (GABA)A receptor α subunits are important for benzodiazepine (BZD) binding and GABA-current potentiation by BZDs, the presence of a γ subunit is required for high affinity BZD effects. To determine which regions unique to the γ2S subunit confer BZD binding and potentiation, we generated chimeric protein combinations of rat γ2S and α1 subunits using a modified protocol to target crossover events to the amino-terminal extracellular region of the subunits. Several chimeras with full open reading frames were constructed and placed into vectors for either voltage-clamp experiments in Xenopus laevisoocytes or radioligand binding experiments in human embryonic kidney 293 cells. Chimeras (χ) containing at least the amino-terminal 161 amino acids of γ2S bound BZDs with wild-type affinity when coexpressed with α1 and β2 subunits. Further analysis of the γ2S binding site region uncovered two areas, γ2S K41-W82 and γ2S R114-D161, that together are necessary and sufficient for high affinity BZD binding. Surprisingly, although the 161-amino acid residue amino terminus of the γ2S subunit is sufficient for high affinity BZD binding, it is not sufficient for efficient allosteric coupling of the GABA and BZD binding sites, as demonstrated by reduced diazepam potentiation of the GABA-gated current and GABA potentiation of [3H]flunitrazepam binding. Thus, by using γ/α chimeras, we identified two γ2 subunit regions required for BZD binding that are distinct from domain or domains responsible for allosteric coupling of the BZD and GABA binding sites.
Footnotes
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Send reprint requests to: Dr. Cynthia Czajkowski, Department of Neurophysiology, University of Wisconsin, 1300 University Avenue, Room 197A MSC, Madison, WI 53706. E-mail:czajkowski{at}neurophys.wisc.edu
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↵1 The affinities for [3H]muscimol binding measured are 2–5-fold lower than values reported by others in the field due to different assay conditions. In our binding assays, incubations with radioligand are carried out at room temperature (as opposed to on ice or 4°) to more closely mimic our electrophysiological recording conditions.
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This work was supported in part by a grant to the University of Wisconsin Medical School under the Howard Hughes Medical Institute Research Program for Medical Schools and by grants to C.C. from The Epilepsy Foundation of America, National Institutes of Health–National Institute of Neurological Disorders and Stroke, and the March of Dimes Birth Defects Foundation. C.C. is a recipient of the Burroughs Wellcome Fund New Investigator Award in the Basic Pharmacological Sciences. Portions of this work have been reported previously in abstract form (Boileau et al., 1996).
- Abbreviations:
- BZD
- benzodiazepine
- GABA
- γ-aminobutyric acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HEK
- human embryonic kidney
- TRCP
- targeted random chimera production
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- Received September 2, 1997.
- Accepted October 24, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
