Structural evidence that propofol stabilizes different GABA(A) receptor states at potentiating and activating concentrations.
The GABA(A) 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 GABA(A) 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 GABA(A) alpha1 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 alpha1 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.
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Daniel B
DB
Williams
Myles H
MH
Akabas
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D019943:Amino Acid Substitution; D018686:Anesthetics, Intravenous / Q000494:pharmacology*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D006993:Hypnotics and Sedatives / Q000494:pharmacology*; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010636:Phenols / Q000494:pharmacology; D015742:Propofol / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D018686:Anesthetics, Intravenous; D006993:Hypnotics and Sedatives; D010636:Phenols; D011963:Receptors, GABA-A; D013439:Sulfhydryl Reagents; C035407:2,6-di-tert-butylphenol; D005680:gamma-Aminobutyric Acid; D002731:4-Chloromercuribenzenesulfonate; D015742:Propofol