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J. Biol. Chem., Vol. 277, Issue 45, 43002-43010, November 8, 2002
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From the Departments of Physiology & Biophysics and of
Neuroscience, Albert Einstein College of Medicine, Bronx, New York
10461
The
GABAA Receptor M2-M3 Loop Secondary Structure and
Changes in Accessibility during Channel Gating*
-aminobutyric acid type A
(GABAA) receptor M2-M3 loop structure and its role
in gating were investigated using the substituted cysteine
accessibility method. Residues from 
1Arg-273 to
1Ile-289 were mutated to cysteine, one at a time.
MTSET+ or MTSES
reacted with all mutants from
1R273C to 1Y281C, except
1P277C, in the absence and presence of GABA. The
MTSET+ closed-state reaction rate was >1000 liters/mol-s
at 1N274C, 1S275C, 1K278C,
and 1Y281C and was <300 liters/mol-s at
1R273C, 1L276C, 1V279C,
1A280C, and 1A284C. These two groups of
residues lie on opposite sides of an -helix. The fast reacting group
lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment -helix extends about two helical turns beyond 1N274 (20'), aligned with the extracellular
ring of charge. At 1S275C, 1V279C,
1A280C, and 1A284C the reaction rate was
faster in the presence of GABA. The reagents had no functional effect
on the mutants from 1A282C to 1I289C,
except 1A284C. Access may be sterically hindered
possibly by close interaction with the extracellular domain. We suggest
that the M2 segment -helix extends beyond the predicted
extracellular end of the M2 segment and that gating induces a
conformational change in and/or around the N-terminal half of the
M2-M3 loop. Implications for coupling ligand-evoked conformational
changes in the extracellular domain to channel gating in the
membrane-spanning domain are discussed.
*
This work was supported by Grants NS30808, GM61925 and
GM63266 from the National Institutes of Health.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 & Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave.,
Bronx, NY 10461. Tel.: 718-430-3360; Fax: 718-430-8819; E-mail:
makabas@aecom.yu.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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