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Vol. 53, Issue 2, 295-303, February 1998
-Aminobutyric AcidA Receptor
Subunits
Department of Neurophysiology, University of Wisconsin-Madison,
Madison, Wisconsin 53706
Although 
-aminobutyric acid (GABA)A receptor 
subunits are important for benzodiazepine (BZD) binding and
GABA-current potentiation by BZDs, the presence of a subunit is
required for high affinity BZD effects. To determine which regions
unique to the 2S subunit confer BZD binding and potentiation, we
generated chimeric protein combinations of rat 2S and 1 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 laevis
oocytes or radioligand binding experiments in human embryonic kidney
293 cells. Chimeras (
) containing at least the amino-terminal 161 amino acids of 2S bound BZDs with wild-type affinity when
coexpressed with 1 and 
2 subunits. Further analysis of the 2S
binding site region uncovered two areas, 2S K41-W82 and 2S
R114-D161, that together are necessary and sufficient for high affinity
BZD binding. Surprisingly, although the 161-amino acid residue amino
terminus of the 2S 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 /
chimeras, we identified two 2 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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