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Subunit Are Crucial for
Benzodiazepine Binding and Allosteric Modulation of
-Aminobutyric AcidA Receptors
Department of Pharmacology and Therapeutics, University of South
Florida College of Medicine, Tampa, Florida 33612 (J.A.),
Children's
Hospital of Philadelphia, Departments of Neurology and Pediatrics,
University of Pennsylvania, Philadelphia, Pennsylvania 19104 (A.B.-K.),
and
Department of Neurobiology, University of Alabama at Birmingham,
Birmingham, Alabama 35294 (D.S.W.)
Benzodiazepines (BZs) exert their therapeutic effects in the mammalian
central nervous system at least in part by modulating the activation of
-aminobutyric acid (GABA)-activated chloride channels. To gain
further insight into the mechanism of action of BZs on GABA receptors,
we have been investigating structural determinants required for the
actions of the BZ diazepam (dzp) on recombinant 
1
22
GABAA receptors. Site-directed mutagenesis was used to
introduce point mutations into the 1 and 2 GABAA receptor subunits. Wild-type and mutant GABAA receptors
were then expressed in Xenopus laevis oocytes or human
embryonic kidney 293 (HEK 293) cells and studied using two-electrode
voltage-clamp and ligand-binding techniques. With this approach, we
identified two tyrosine residues on the 1 subunit (Tyr159 and
Tyr209) that when mutated to serine, dramatically impaired modulation
by dzp. The Y209S substitution resulted in a >7-fold increase in the
EC50 for dzp, and the Y159S substitution nearly abolished
dzp-mediated potentiation. Both of these mutations abolished binding of
the high affinity BZ receptor antagonist [3H]Ro 15-1788
to GABAA receptors expressed in HEK 293 cells. These tyrosine residues correspond to two tyrosines of the 2 subunit (Tyr157 and Tyr205) previously postulated to form part of the GABA-binding site. Mutation of the corresponding tyrosine residues on
the 2 subunit produced only a slight increase in the
EC50 for dzp (~2-fold) with no significant effect on the
binding affinity of [3H]Ro 15-1788. These data suggest
that Tyr159 and Tyr209 of the 1 subunit may be components of the
BZ-binding site on 122 GABAA receptors.
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