Mapping general anesthetic binding site(s) in human α1ß3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue.
Biochemistry
; 51(4): 836-47, 2012 Jan 31.
Article
en En
| MEDLINE
| ID: mdl-22243422
The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [³H]azietomidate photolabeling of αMet-236 and ßMet-286 in the αM1 and ßM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the ß and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [³H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1ß3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [³H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner ß3Val-290, in the ß3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]azietomidate, while no photolabeling of amino acids in the αM2 and ßM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of ß3Met-227 in ßM1 by [³H]TDBzl-etomidate and [³H]azietomidate also provides evidence of a homologous etomidate binding site at the ß3-ß3 subunit interface in the α1ß3 GABA(A)R.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Receptores de GABA-A
/
Anestésicos Generales
/
Etiquetas de Fotoafinidad
/
Subunidades de Proteína
/
Diazometano
/
Etomidato
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Biochemistry
Año:
2012
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos