Numerous classes of general anesthetics inhibit etomidate binding to gamma-aminobutyric acid type A (GABAA) receptors

Abstract

Enhancement of gamma-aminobutyric acid type A receptor (GABA(A)R)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABA(A)R function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABA(A)R protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABA(A)R transmembrane domain at the beta-alpha subunit interface; the etomidate analog [(3)H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, alpha1Met-236 in the M1 helix and betaMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605). Here, we use [(3)H]azietomidate photolabeling of bovine brain GABA(A)Rs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of alpha1Met-236 and betaMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABA(A)R: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate.

FIGURE 1.

Chemical structures of general anesthetics.

FIGURE 2.

Concentration-dependent enhancement of [³H]muscimol binding to purified bovine GABA_AR by propofol (A), pentobarbital (B), isoflurane (C), and n-octanol (D). Plotted are the percentage changes of specific [³H]muscimol binding (5 nm) (mean ± S.E.) in the presence of increasing concentrations of compounds. Binding assays were performed as described (14).

FIGURE 3.

Propofol inhibits [³H]azietomidate photolabeling of the GABA_AR. A, the GABA_AR was photolabeled with 1 μm [³H]azietomidate in the presence of 1 mm GABA, 10 mm clorazepate, and propofol at 0 (□), 1 μm (▵), 10 μm (◇), 100 μm (▿), and 200 μm (○), and covalent ³H incorporation was analyzed by SDS-PAGE. B, shown is the concentration dependence of propofol inhibition, determined by normalizing the ³H in the GABA_AR subunit gel bands in the presence of propofol to that in the absence of propofol. The data points are the average and range from two independent photolabeling experiments. The solid lines are fits of the data to a logistic function. ■, in the same experiment, 200 μm etomidate inhibited photolabeling by ∼90%. Error bars indicate S.E. C and D, propofol inhibits [³H]azietomidate photolabeling of the GABA_AR at both α1Met-236 (αM1) and βMet-286 (βM3). ³H (○, ●) and phenylthiohydantoin-amino acids (□, ■) released during Edman sequencing of fractions enriched in αM1 (C) and βM3 (D) were isolated by SDS-PAGE and reversed-phase HPLC from affinity-purified GABA_AR photolabeled with 0.7 μm [³H]azietomidate in the presence of 1 mm GABA and 10 mm clorazepate with (●, ■) or without (○, □) 150 μm propofol (supplemental Figs. S1–S3). C, sequence analysis of photolabeling in αM1. The peptide beginning at α1Ile-223 (−propofol, initial amount (I₀) = 0.6 pmol, repetitive yield (R) = 91%; +propofol, I₀ = 1.6 pmol, R = 83%) was the only peptide that persisted after treatment with o-phthalaldehyde prior to cycle 11 to prevent sequencing of any peptide not containing a proline at this cycle. Release of ³H in cycle 14 established photolabeling of α1Met-236 (−propofol) at 140 cpm/pmol (95–210 cpm/pmol) that was reduced by propofol to 35 cpm/pmol (22–56 cpm/pmol). These ranges were calculated using the standard errors from the fits for R and I₀. D, sequence analysis of photolabeling in βM3. The peptide beginning at βAla-280 was present in both samples (−propofol, I₀ = 0.7 pmol, R = 93%; +propofol, I₀ = 0.5 pmol, R = 96%), and the peak of release of ³H in cycle 7 indicates photolabeling of βMet-286 (−propofol) at 50 cpm/pmol (33–79 cpm/pmol) that was reduced by propofol to 19 cpm/pmol (10–39 cpm/pmol). E, shown is the alignment of subtypes of α or β subunits in the regions of M1 or M3 (both in gray), respectively, illustrating the high sequence conservation in these regions. In boldface are the labeled Met residues as well as the conserved Pro residue in cycle 11 of Edman degradation (C).

FIGURE 4.

[³H]Azietomidate photolabeling of the GABA_AR is inhibited by bioactive (GABA-enhancing) barbiturates (pentobarbital (A), phenobarbital (B), and (−)-methylphenobarbital (C)), but not by an inactive stereoisomer ((+)-methylphenobarbital (D)). The GABA_AR was photolabeled with 1 μm [³H]azietomidate in the presence of 1 mm GABA, 10 mm clorazepate, and concentrations of barbiturates, and covalent ³H incorporation was analyzed by SDS-PAGE. The concentration dependence of inhibition was determined by normalizing the ³H in the GABA_AR subunit gel bands in the presence of barbiturate to that in the absence.

FIGURE 5.

Modulation of [³H]azietomidate photolabeling by volatile anesthetics and alcohols. A, the GABA_AR was photolabeled with 1 μm [³H]azietomidate in the presence of 1 mm GABA, 10 mm clorazepate, and isoflurane at 0 (□), 100 μm (▵), 1 mm (◇), 3 mm (▿), and 10 mm (○), and covalent ³H incorporation was analyzed by SDS-PAGE. B, the concentration dependence of isoflurane inhibition was determined by normalizing the ³H in the GABA_AR subunit gel bands in the presence of isoflurane to that in the absence. The solid line is a fit of the data to a logistic function. C and D, ethanol and octanol did not modulate [³H]azietomidate photolabeling of GABA_AR subunits.