title: Benzodiazepines induce a conformational change in the region of the gamma-aminobutyric acid type A receptor alpha(1)-subunit M3 membrane-spanning segment.
abstract: Benzodiazepine binding to gamma-aminobutyric acid type A (GABA(A)) receptors allosterically modulates GABA binding and increases the currents induced by submaximal GABA concentrations. Benzodiazepines induce conformational changes in the GABA-binding site in the extracellular domain, but it is uncertain whether these conformational changes extend into the membrane-spanning domain where the channel gate is located. Alone, benzodiazepines do not open the channel. We used the substituted-cysteine-accessibility method to investigate diazepam-induced conformational changes in the region of the alpha(1)-subunit M3 membrane-spanning segment. In the absence of diazepam or GABA, pCMBS(-) did not react at a measurable rate with cysteine-substitution mutants between alpha(1)Phe296 and alpha(1)Glu303. In the presence of 100 nM diazepam, pCMBS(-) reacted with alpha(1)F296C, alpha(1)F298C, and alpha(1)L301C but not with the other cysteine mutants between alpha(1)Phe296 and alpha(1)Glu303. These three mutants are a subset of the five residues that we previously showed reacted with pCMBS(-) applied in the presence of GABA. The pCMBS(-) reaction rates with these three cysteine mutants were similar in the presence of diazepam and GABA. Thus, diazepam, which binds to the extracellular domain, induces a conformational change in the membrane-spanning domain that is similar to a portion of the change induced by GABA. Because diazepam does not open the channel, these results provide structural evidence that the diazepam-bound state represents an intermediate conformation distinct from the open and resting/closed states of the receptor. The diazepam-induced conformational change in the M3 segment vicinity may be related to the mechanism of allosteric potentiation.
authors.affiliation: Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University College of Physicians and Surgeons, New York, New York, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Williams
authors.forename: M H
authors.initials: MH
authors.lastname: Akabas
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology*; D002243:Carbolines / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003975:Diazepam / Q000494:pharmacology; D005442:Flumazenil / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D002243:Carbolines; D018757:GABA Modulators; D008565:Membrane Proteins; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005442:Flumazenil; D002731:4-Chloromercuribenzenesulfonate; C024915:beta-carboline-3-carboxylic acid ethyl ester; D003545:Cysteine; D003975:Diazepam