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J. Biol. Chem., Vol. 278, Issue 15, 13166-13172, April 11, 2003
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From the Department of Physiology, University of Wisconsin-Madison,
Madison, Wisconsin 53706
The GABA-binding site undergoes structural
rearrangements during the transition from agonist binding to channel
opening. To define possible roles of the GABAA
receptor
The GABAA Receptor
1 Subunit
Pro174-Asp191 Segment Is Involved in GABA
Binding and Channel Gating*
1 subunit
Pro174-Asp191 segment in these processes, we
used the substituted cysteine accessibility method to
characterize this region. Each residue was individually mutated to
cysteine, expressed with wild-type 
2 subunits in
Xenopus laevis oocytes, and examined using
two-electrode voltage clamp. Most mutations did not alter GABA
EC50 values. The D183C mutation produced a 7-fold reduction
in GABA sensitivity. There were no significant changes in the
KI values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl
methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-,
A181C-, D183C-, R186C- and N188C-containing receptors. The pattern of
accessibility suggests that this protein segment is aqueous-exposed and
adopts a random coil conformation. Both GABA and SR-95531 slowed
covalent modification of V178C, V180C, and D183C, indicating that these residues may line the GABA-binding site. Further,
pentobarbital-induced channel activation accelerated modification of
V180C and A181C and slowed the modification of R186C, suggesting that
this region of the 1 subunit may act as a dynamic
element during channel-gating transitions.
*
This work was supported by NINDS, National Institutes of
Health Grant 34727 (to C. C.) and a postdoctoral fellowship (to
J. G. N.) from the Natural Sciences and Engineering Research Council of Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology,
Rm. 197 MSC, University of Wisconsin-Madison, 1300 University Ave.,
Madison, WI 53706. Tel.: 608-265-5863; Fax: 608-265-5512; E-mail:
czajkowski@physiology.wisc.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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