Mapping General Anesthetic Binding Site(s) in Human α1β3 γ-Aminobutyric Acid Type A Receptors with [³H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue

Abstract

The γ-aminobutyric acid type-A receptor (GABAAR) 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 GABAARs purified from bovine brain, [3H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABAAR transmembrane domain at the interface between the β and α subunits. To further define GABAAR 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-α1β3 GABAARs and obtain a more extensive identification of photolabeled GABAAR amino acids. [3H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [3H]azietomidate, while no photolabeling was detected of amino acids in the αM2 or βM2 helices that also border the etomidate binding site. The location of these photolabeled amino acids in GABAAR homology models derived from the recently solved structures of prokaryote (GLIC) or invertebrate (GluCl) homologs 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 GABAAR intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [3H]TDBzl-etomidate and [3H]azietomidate also provides evidence of an homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABAAR.

Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor*

“…Saccharomyces aureus endoproteinase Glu-C (EndoGlu-C) and Lysobacter enzymogenes endoproteinase Lys-C (EndoLys-C) were from Worthington and Roche Applied Science, respectively. Purification of Human ␣1␤3␥2 GABA A R-␣1␤3␥2 GABA A Rs with a FLAG epitope at the N terminus of the ␣1 subunit were expressed in a tetracycline-inducible, stably transfected HEK293S cell line and purified on an anti-FLAG affinity resin with modifications of procedures used to purify a previously characterized tetracycline-inducible FLAG-␣1␤3 GABA A R (15,20 (14). The total concentration of sites was determined at 500 nM [ 3 H]muscimol and with 1 mM GABA to determine nonspecific binding.…”

“…The total concentration of sites was determined at 500 nM [ 3 H]muscimol and with 1 mM GABA to determine nonspecific binding. Anesthetic modulation of 2-3 nM [ 3 H]muscimol binding was measured as described (15,20), except that samples were incubated for 60 min at room temperature before addition of polyethylene glycol and ␥-globulins and then filtered after a 30-min incubation at room temperature. The modulation results are presented as the percentage of the specifically bound [ 3 H]muscimol over that without modulators.…”

“…For chemical cleavage at the C terminus of methionines, samples immobilized on PVDF sequencing filters were treated with cyanogen bromide as described (21,22). For chemical cleavage at the C terminus of tryptophans, samples on PVDF filters were treated with BNPS-skatole as described (23), except that after precipitation of the excess BNPS-skatole, the digestion solution was loaded onto a second PVDF filter, and material on the two filters was sequenced simultaneously (15).…”

“…Molecular Modeling-Comparison of structural models for ␣1␤3 GABA A R constructed by homology with GLIC and GluCl (15) established that the positions of amino acids of ␤M3/␣M1 and ␣M3/␤M1 contributing to the ␤ ϩ -␣ Ϫ and ␣ ϩ -␤ Ϫ interfaces, respectively, were the same. Compared with the GLICderived structure, there was an increased distance in the GluCl structure between the M3 and M1 helices where the allosteric potentiator ivermectin is bound in GluCl.…”

“…In the transmembrane domain, M2 helices from each subunit associate around a central axis to form the ion channel, and amino acids from the M1 and M3 helices of adjacent subunits contribute to the subunit interfaces. The etomidate-binding sites, identified by photoaffinity labeling of amino acids in ␤M3 and ␣M1, are in the two ␤ ϩ -␣ Ϫ subunit interfaces about 50 Å below the agonist sites (14,15). We reported recently that the R-enantiomer of 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB) is an extremely potent, photoreactive barbiturate that rivals etomidate in potency and stereoselectivity (16).…”