Identification of Anesthetic Binding Sites on Human Serum Albumin Using a Novel Etomidate Photolabel

Abstract

We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(؉) enantiomer being significantly more potent than the S(؊) enantiomer. Its effects on ␣1␤2␥2s GABA A receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (␤2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds ϳ75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABA A receptor or other putative targets.Although it is now widely accepted that general anesthetics exert their effects by binding directly to their protein targets (1-3), information on the precise molecular locations of these binding sites has been slow in coming. Most information has been derived from in vitro electrophysiological experiments in which putative anesthetic targets, ion channels, or receptors, are genetically modified. What this approach provides is information on the molecular determinants of anesthetic sensitivity, but it is usually impossible to tell whether these determinants represent portions of anesthetic binding sites or regions of the channel or receptor that are responsible for transducing anesthetic binding into changes in channel gating. In principle, the most direct approach would be to determine a high resolution crystal structure of the ion channel or receptor in question in the presence and absence of anesthetics. Unfortunately, because of the difficulties in crystallizing membrane proteins, such experiments are still some way off. An alternative strategy that has recently had some success is to use anesthetics that have been modified so that they contain photoactivatable groups. When these anesthetic analogs are illuminated with a bright light, they are converted into highly reactive intermediates, which then, hopefully, bind irreversibly to their targets.The idea of using photoaffinity labeling to identify anesthetic binding sites was first implemented using the volatile anesthetic halothane (4, 5). Halothane ...

A propofol binding site on mammalian GABAA receptors identified by photolabeling

“…We exposed HSA to light with wavelengths >320 nm in the absence (control) or presence of either the protiated or deuterated forms of ortho -propofol diazirine, then analyzed tryptic digests of the samples using LC-MS 26 . In the control samples, tandem spectra (MS2) unambiguously identified peptides that contained 569 of the 585 amino acids in HSA (97% coverage; Supplementary Fig.…”

“…GABA A receptors formed from either α 1 β 2 γ 2s or α 1 β 3 subunits were transiently expressed in HEK 293 cells and currents were recorded using whole-cell patch clamping as described previously 26 . Extracellular solution (140 mM NaCl, 2.5 mM KCl, 2 mM MgCl 2 , 10 mM HEPES, 10 mM D-glucose, 1 mM CaCl 2 , pH 7.4) was used to perfuse the cells.…”

“…The sample was then continuously stirred while being irradiated for 120 seconds with light from a mercury-xenon arc lamp whose light had been filtered by reflection from a dichroic mirror (to remove the infrared) and by passing through an interference filter (λ > 320 nm). After photolabeling, the protein was denatured with guanidine hydrochloride, the di-sulfide bonds were reduced using dithiothreitol and their reformation blocked using iodoacetamide, and then the protein was digested with trypsin following dialysis 26 . The resulting peptides were then separated using HPLC and analyzed using mass spectrometry as previously described 26 .…”

“…After photolabeling, the protein was denatured with guanidine hydrochloride, the di-sulfide bonds were reduced using dithiothreitol and their reformation blocked using iodoacetamide, and then the protein was digested with trypsin following dialysis 26 . The resulting peptides were then separated using HPLC and analyzed using mass spectrometry as previously described 26 .…”

Clinical and Molecular Pharmacology of Etomidate

“…Husain and colleagues synthesized a diaziryl derivative, azi-etomidate 3 , while Bright et al 134 produced an azide. These photolabels display stereoselectivity and pharmacological activity almost identical to that of etomidate in both animals and GABA A receptors.…”