TY - JOUR
T1 - Structural evidence that propofol stabilizes different GABAA receptor states at potentiating and activating concentrations
AU - Williams, Daniel B.
AU - Akabas, Myles H.
PY - 2002/9/1
Y1 - 2002/9/1
N2 - 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 α1 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 α1 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.
AB - 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 α1 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 α1 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.
KW - Acetylcholine
KW - Anesthesia
KW - Benzodiazepine
KW - Ethanol
KW - Glycine
KW - Isoflurane
UR - http://www.scopus.com/inward/record.url?scp=0036759617&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036759617&partnerID=8YFLogxK
U2 - 10.1523/jneurosci.22-17-07417.2002
DO - 10.1523/jneurosci.22-17-07417.2002
M3 - Article
C2 - 12196563
AN - SCOPUS:0036759617
SN - 0270-6474
VL - 22
SP - 7417
EP - 7424
JO - Journal of Neuroscience
JF - Journal of Neuroscience
IS - 17
ER -