Mapping general anesthetic binding site(s) in human alpha1beta3 gamma-aminobutyric acid type A receptors with [(3)H]TDBzl-etomidate, a photoreactive etomidate analogue.
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Chiara DC, Dostalova Z, Jayakar SS, Zhou X, Miller KW, Cohen JB
Mapping general anesthetic binding site(s) in human alpha1beta3 gamma-aminobutyric acid type A receptors with [(3)H]TDBzl-etomidate, a photoreactive etomidate analogue.
Biochemistry. 2012 Jan 31;51(4):836-47. doi: 10.1021/bi201772m. Epub 2012 Jan 23.
- PubMed ID
- 22243422 [ View in PubMed]
- Abstract
The gamma-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, [(3)H]azietomidate photolabeling of alphaMet-236 and betaMet-286 in the alphaM1 and betaM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the beta and alpha subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [(3)H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-alpha1beta3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [(3)H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner beta3Val-290, in the beta3M3 transmembrane helix, as well as alpha1Met-236 in alpha1M1, a residue photolabeled by [(3)H]azietomidate, while no photolabeling of amino acids in the alphaM2 and betaM2 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 [(3)H]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 beta3Met-227 in betaM1 by [(3)H]TDBzl-etomidate and [(3)H]azietomidate also provides evidence of a homologous etomidate binding site at the beta3-beta3 subunit interface in the alpha1beta3 GABA(A)R.