A propofol binding site on mammalian GABAA receptors identified by photolabeling

Abstract

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA (γ-aminobutyric acid type A) receptors, but where it binds to this receptor is not known and has been a matter of some debate. We synthesized a new propofol analog photolabeling reagent whose biological activity is very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin that have been identified using X-ray crystallography. Using a combination of protiated and deuterated versions of the reagent to label mammalian receptors in intact membranes, we identified a new binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit at the interface between the transmembrane domains and the extracellular domain and lies close to known determinants of anesthetic sensitivity in the transmembrane segments TM1 and TM2.

Figure 1. Characterization of ortho-propofol diazirine

(a) The chemical structures of propofol (left) and ortho-propofol diazirine (1). (b) The percentage potentiation of GABA-evoked currents by propofol (3 μM; n=12), ortho-propofol diazirine (5 μM: n=12), para-methylene-propofol diazirine (4; 50 μM; n=7) and meta-propofol diazirine (2; 200 μM; n=3). GABA (3 μM; EC₇) was applied to HEK293 cells expressing α₁β₂γ_2s GABA_A receptors, and the current was recorded under whole-cell patch clamp. The inset shows a typical current trace during switches into GABA, in the presence and absence of 5 μM ortho-propofol diazirine. (c) UV absorption spectrum of ortho-propofol diazirine in ethanol (0.2 mg ml⁻¹). (d) Inhibition of [³⁵S]-TBPS binding to GABA_A β₃ homomers by ortho-propofol diazirine. The IC₅₀ concentration for inhibition of TBPS binding was 2.9 ± 0.4 μM (n=3). (e) The percentage potentiation of GABA-evoked currents by ortho-propofol diazirine acting on α₁β₃ GABA_A receptor heteromers. The EC₅₀ concentration for potentiation was 1.7 ± 0.7 μM (n=10) with a Hill coefficient of 0.8±0.2 and the maximum potentiation was 151 ± 18%. (The point at the highest drug concentration was excluded from the fit to the Hill equation.) (f) Quantal dose-response curves for propofol and ortho-propofol diazirine for loss of righting reflex in rats. The ED₅₀ concentrations for propofol and ortho-propofol diazirine were 4.7 ± 0.8 mg kg⁻¹ (n=12) and 14.7 ± 0.2 mg kg⁻¹ (n=13) respectively. All error bars are standard errors of the mean and if not shown, were smaller than the size of the symbols.

Figure 2. Labeling human serum albumin with ortho-propofol diazirine

Calculated and observed isotopic distributions for (a) unlabeled YTK peptide, (b) YTK peptide labeled with protiated ortho-propofol diazirine and (c) YTK peptide labeled with partially deuterated ortho-propofol diazirine. MS2 data for peptides YTK (d) and HKPK (e) with protiated ortho-propofol diazirine (black) and deuterated ortho-propofol diazirine (red). The asterisks denote the presence of photolabel bound to the peptide fragment. The two sites are illustrated in (f) with PR1 being in subdomain IIIA (dark blue) and PR2 being in subdomain IIIB (light blue). More detailed views are shown in (g) for PR1 where the labeled Y411 is shown and in (h) for PR2 where the labeled H535 is shown. Dashed lines mark the distances between the propofol molecules to Y411 (4.9 A) and to H535 (5.0 A).

Figure 3. Labeling GABA_A receptors with ortho-propofol diazirine

With both α₁β₃ heteromers and β₃ homomers only a single amino acid, H267, was labeled. (a) MS2 data for the peptide 260-TMTTINTHL-268 labeled with the protiated ortho-propofol diazirine (black) and the deuterated ortho-propofol diazirine (red) superimposed. (b) Fragmentation diagram showing that the MS2 data are consistent with the ortho-propofol diazirine labeling the histidine side chain which subsequently rearranges as internal ions H* and TH* following fragmentation. (c) A view of our proposed propofol binding site seen from the center of the pore. Two neighboring subunits are shown in yellow and blue and a single ortho-propofol diazirine molecule is shown associated with the yellow subunit. (d) A view from the extracellular side of the receptor, but with the extracellular domain removed for clarity. The β₃ homopentamer is shown with five equivalent ortho-propofol diazirine molecules bound. (e) A surface representation of our proposed propofol binding pocket with an ortho-propofol diazirine molecule sitting in the pocket close to H267 which is the amino acid that is photolabeled. (f) Electrophysiological data from α₁β₃ heteromers showing the effects of the mutations β₃-H267A and β₃-F221W on the GABA apparent affinity (n=21), (g) the extent to which 3 μM ortho-propofol diazirine potentiates a GABA-evoked chloride current (at the GABA EC₆₀) (n=4–10) and (h) the extent to which 10 μM ortho-propofol diazirine directly activates the receptor (n=4). All error bars are standard errors of the mean.