Molecular Dissection of Benzodiazepine Binding and Allosteric Coupling Using Chimeric γ-Aminobutyric AcidA Receptor Subunits

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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.

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