2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate:  A Derivative of the Stereoselective General Anesthetic Etomidate for Photolabeling Ligand-Gated Ion Channels

Abstract

To locate general anesthetic binding sites on ligand-gated ion channels, a diazirine derivative of the potent intravenous anesthetic, R-(+)-etomidate (2-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate), has been synthesized and characterized. R-(+)-Azietomidate [2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate] anesthetizes tadpoles with an EC(50) of 2.2 microM, identical to that of R-(+)-etomidate. At this concentration both agents equally enhanced GABA-induced currents and decreased binding of the caged-convulsant [(35)S]TBPS to GABA(A) receptors. In all of the above actions R-(+)-azietomidate is about an order of magnitude more potent than S-(-)-azietomidate, an enantioselectivity comparable to etomidate's. R-(+)-Azietomidate also inhibits acetylcholine-induced currents in nicotinic acetylcholine receptors, with about twice the potency of the parent compound. [(3)H]Azietomidate photoincorporated into Torpedo nicotinic acetylcholine receptor-rich membranes. Desensitization decreased photoincorporation into the delta-subunit and increased that into the alpha-subunit. The latter increase was confined to a proteolytic fragment containing the first three transmembrane segments. Thus, R-(+)-azietomidate is a potent stereoselective general anesthetic and an effective photolabel.

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

“…Materials-Nonradioactive AziPm was synthesized as described (21) (26), which was also resynthesized at 19 Ci/mmol by catalytic reduction of m-bromoazietomidate with tritium gas. o-PD was synthesized from 2-isopropyl-6-trifluoroacetylphenol as described (20) with the purity Ͼ96% as judged by 1 H and 19 F NMR.…”

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABAA and serotonin receptors

“…However, in 11.5 mM cholate and 0.86 mM asolectin, higher etomidate concentrations were required to cause enhancement, possibly because the higher lipid concentration depleted the free concentration of etomidate, which is very lipid soluble. 47 This factor should decline in importance as the concentration of etomidate increases, and, indeed, at !30 lM etomidate enhancement was similar in all samples. When compared at 10 lM etomidate, however, enhancement was somewhat lower in the reconstituted samples than in the membranes (P ¼ 0.033, unpaired t-test).…”

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

“…GABA potentiation and direct activation share the same dependence on ␤ subunit isoforms, the same stereoselectivity for etomidate (Tomlin et al, 1998) [and azietomidate (Husain et al, 2003)], and parallel responses for substitutions at ␤M2-15 Jurd et al, 2003). In addition, for wild-type GABA A R, concentrations of etomidate up to 100 M produce a progressive decrease of GABA EC 50 values without evidence of saturation, and the data were well-fit by an allosteric model with each GABA A R containing two equivalent etomidate binding sites contributing to direct gating and agonist potentiation (Rusch et al, 2004).…”

“…3 H]azietomidate (11 Ci/mmol) was synthesized as described previously (Husain et al, 2003). (Sources of antibodies and reagents are described in the supplemental Materials and Methods, available at www.jneurosci.org as supplemental material.…”

“…Photoaffinity labeling, which provides an experimental approach to directly identify amino acids contributing to a drug binding site without previous assumptions about the protein points of contact (Kotzyba-Hibert et al, 1995), has been used extensively to identify amino acids contributing to ligand binding sites in the Torpedo nAChR (Mourot et al, 2006) and for GABA A R agonists and benzodiazepines (Smith and Olsen, 1994;Duncalfe et al, 1996;Sawyer et al, 2002). We now use 2-(3-methyl-3H-diaziren-3-yl) ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate ([ 3 H]azietomidate), a photoreactive analog of etomidate that retains anesthetic and GABA A R modulatory properties (Husain et al, 2003), to provide the first identification of amino acids contributing to an anesthetic binding site in a GABA A R. The two labeled residues, ␣M1-14 (␣Met-236) and , are shown by homology modeling to border a pocket in the TMD located at the same subunit interfaces that contain the GABA binding site in the extracellular domain. The ␣M1 helix has not been implicated previously in etomidate function, whereas ␤M3-4 is an anesthetic sensitivity determinant (Mihic et al, 1997;Krasowski et al, 1998;Bali and Akabas, 2004).…”