Identification of a GABA_AReceptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

Abstract

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA A Rs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([ 3 H]azietomidate), which retains anesthetic potency in vivo and enhances GABA A R function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA A R anesthetic binding site. For GABA A Rs purified by affinity chromatography from detergent extracts of bovine cortex, [ 3 H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC 50 ϭ 30 M). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the ␣M1 transmembrane helix at ␣1Met-236 (and/or the homologous methionines in ␣2,3,5), not previously implicated in etomidate function, and one within the ␤M3 transmembrane helix at ␤3Met-286 (and/or the homologous methionines in ␤1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between ␤ and ␣ subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket ϳ50 Å below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

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

“…2C) established that the 56-kDa band also contained primarily the ␣1 subunit, and the 59-and 61-kDa bands contained primarily the ␤3 subunit, although the ␥2 subunit (beginning at Lys-2) was distributed in all three bands. 3 H]azietomidate-photolabeled ␣1␤3␥2 GABA A R, sequence analysis of subunit fragments isolated from digests enriched in ␣1 or ␤3 subunits identified etomidate-inhibitable photolabeling of the same amino acids (␣1Met-236 in ␣M1 and ␤3Met-286 in ␤M3) as in the GABA A R purified from bovine brain (14) or in the ␣1␤3 GABA A R (data not shown) (15).…”

“…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 ␥2 subunit is distributed diffusely in all three bands. 3 H]mTFD-MPAB (0.6 M) on a preparative scale in the absence or presence of 1 mM pentobarbital, and we used previously developed strategies (14,15) to identify photolabeling within each of the four transmembrane helices of the ␤3 subunit, the subunit with the highest 3 H incorporation. When an EndoLys-C digest of material enriched in ␤3 subunits (eluted from the 59-and 61-kDa gel bands) was fractionated by reversed-phase HPLC (Fig.…”

“…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).…”

Structural and Functional Studies of the Modulator NS9283 Reveal Agonist-like Mechanism of Action at α4β2 Nicotinic Acetylcholine Receptors

“…A classic example of this is etomidate and benzodiazepine modulators at GABA A Rs, which work through different binding sites and have very distinct modulatory profiles (11). Etomidate binds to sites in the transmembrane region of the receptor where it modulates GABA A R efficacy levels at low concentrations and directly activates at high concentrations, similarly to the barbiturate type of modulators at this receptor (12,13). In contrast, benzodiazepines only modulate the GABA potency and are known to bind in an extracellular pocket in the receptor ␣-␥ interface, a pocket that is structurally similar to the GABA-binding ␤-␣-subunit pocket (6,9).…”