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Vol. 57, Issue 3, 474-484, March 2000
-Aminobutyric Acid Type A Receptor
2 Subunit Affects Allosteric Sensitivity to GABA and
Anesthetics
Department of Pharmacology, The Royal Danish School of Pharmacy,
Copenhagen, Denmark (B.X.C., A.C.E., U.K., A.S.); and Department of
Molecular and Medical Pharmacology, UCLA School of Medicine, Los
Angeles, California (R.W.O)
Site-directed mutagenesis of the 
-aminobutyric acid type A
(GABAA) receptor 
2 subunit has demonstrated
that conversion of a conserved glycine residue located at the entrance
to the first transmembrane domain into the homologous 
1
residue phenylalanine alters the modulating effects of four different
i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol.
Using the baculovirus expression system in Spodoptera
frugiperda 9 cells, anesthetic-induced enhancement of
[3H]muscimol and [3H]flunitrazepam binding
in receptors containing the 2(G219F) point mutation
displayed a significantly reduced efficacy in modulation by all four
i.v. anesthetics tested. Furthermore, GABAA receptors containing the 
1(G223F) point mutation also
significantly decreased the maximal effect of etomidate- and
propofol-induced enhancement of ligand binding. Conversely, the
homologous point mutation in 1 receptors (F261G) changed
the i.v. anesthetic-insensitive receptor to confer anesthetic
modulation of [3H]muscimol binding. Consistent with the
binding, functional analysis of pentobarbital-enhanced GABA currents
recorded with whole-cell patch clamp demonstrated the
2(G219F) subunit mutation eliminated the potentiating
effect of the anesthetic. Similarly, propofol-enhanced GABA currents
were potentiated less in
12(G219F)2 receptors than
in 122 receptors. Although
ligand binding displayed comparable KD values for muscimol
among wild-type, 122, and
mutant receptors, patch-clamp recordings showed that
12(G219F)2 receptors had a
significantly more potent response to GABA than did
122 or 1(G223F)22. The
12(G219F)2 receptors also
were more sensitive to direct channel activation by pentobarbital and
propofol in the absence of GABA. These results suggest that the first
transmembrane glycine residue on the 2 subunit may be
important for conformational or allosteric interactions of channel
gating by both GABA and anesthetics.
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