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The Journal of Neuroscience, September 1, 2002, 22(17):7417-7424
Structural Evidence that Propofol Stabilizes Different
GABAA Receptor States at Potentiating and Activating
Concentrations
Daniel B.
Williams1, 3 and
Myles H.
Akabas1, 2
Departments of 1 Physiology and Biophysics and
2 Neuroscience, Albert Einstein College of Medicine, Bronx,
New York 10461, and 3 Integrated Program in Cellular,
Molecular, and Biophysical Studies, Columbia University, New York, New
York 10032
The GABAA receptor is a target of many general
anesthetics, such as propofol. General anesthetic binding sites are
distinct from the GABA binding sites. At low concentrations, the
anesthetics potentiate the currents induced by submaximal GABA
concentrations. At higher concentrations the anesthetics directly
activate GABAA receptors. In contrast, benzodiazepines,
such as diazepam, only potentiate currents induced by submaximal GABA
concentrations. Channel kinetic studies suggest that these drugs
stabilize different receptor states. We previously showed that the
accessibility of the anionic sulfhydryl reagent
p-chloromercuribenzenesulfonate (pCMBS ) applied extracellularly to cysteines
substituted for residues in the GABAA  1
subunit M3 membrane-spanning segment was state-dependent. The subset of
pCMBS-accessible, M3 segment cysteine mutants acts
as a reporter for receptor conformation. Here we show that
pCMBS, applied in the presence of a potentiating
concentration of propofol, reacts with a subset of 1
subunit, M3 segment, cysteine-substitution mutants (Y294C, V297C,
I302C, F304C). In the presence of a directly activating concentration
of propofol pCMBS reacts with a different subset
of the M3 cysteine-substitution mutants (Y294C, S299C, I302C, E303C,
A305C). These subsets are distinct from the subsets of M3
cysteine-substitution mutants that are reactive with
pCMBS in the absence and presence of GABA and in
the presence of diazepam. We hypothesize that distinct subsets of
reactive residues represent distinct conformations or ensembles of
conformations of the receptor. These results provide structural
evidence for at least five distinct receptor states, three
nonconducting states, resting, diazepam-bound and potentiating
propofol-bound, and two conducting-desensitized states, the activating
propofol-bound and GABA-bound states.
Key words:
acetylcholine; glycine; anesthesia; benzodiazepine; isoflurane; ethanol
Copyright © 2002 Society for Neuroscience 0270-6474/02/22177417-08$05.00/0
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