Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol

Abstract

Background: Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the "+" of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the "-" side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy.

Method: We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the β subunit on activation of the α1β3 GABAA receptor by propofol.

Results: We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol.

Conclusion: We conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.

Fig. (1)

Top view of the β3 homomeric (left) and α1β3 heteromeric (right) GABA_A receptor. The putative propofol binding sites in the β3 receptor are located at each of the five β-β interfaces, predominantly in the subunit contributing the “-” side of the interface. Tryptophan-substitutions of Y143, F221, and Q224 drastically reduce or abolish activation by propofol. We propose that in the α1β3 receptor, the propofol binding sites are located at the β-β and α-β where Y143, F221, and Q224 control drug interactions with the receptors, and at the β-α interfaces where the M286 defines the putative binding site.

Fig. (2)

Graphic presentation of estimation of channel open probability. To estimate channel open probability for a given agonist, we first determined P_o of spontaneously active receptors (P_o,spont^est) and conditions required to attain the maximal open probability (P_o^est of 1). The current level corresponding to estimated open probability of 0 was attained by exposing receptors to 100 µM picrotoxin (PTX). The current level corresponding to P_o^est of 1 was obtained by activating receptors with a saturating concentration of GABA in the presence of 100 µM pentobarbital (GABA + PB). All other current levels, including the holding current and the peak current in the presence of GABA or propofol, were compared to this range to obtain estimates of P_o,spont, and P_o in the presence of various concentrations of GABA or propofol. For illustrative purposes, the data traces were obtained from different receptors (block by picrotoxin from α1β3(M286W+F221W), potentiation by pentobarbital from α1β3 wild-type). The human α1β3 GABA_A receptors were expressed in Xenopus oocytes. Harvesting of oocytes was conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The protocol was approved by the Animal Studies Committee of Washington University in St. Louis. Current traces were recorded using standard two-electrode voltage clamp as described previously [24].

Fig. (3)

Functional results from studies of α1β3 GABA_A receptors. The data points show averaged values for estimated open probability (P_o^est) of wild-type and mutant α1β3 receptors activated by propofol (A) or GABA (B). The P_o^est values were obtained as described in Fig. 2. The curves were generated by fitting the following equation:

Fig. (4)

Predicted propofol binding sites at intersubunit interfaces in an α1β3 GABA_A receptor homology model. The figure shows views from the top with pore at the bottom (left panels) and sideviews from the pore for β-β (A), α-β (B), and β-α (C) interfaces (right panels). Note that the interfaces are referred to with the subunit providing the “+” side of the interface named first. The α1 subunit is shown in blue and the β3 subunits are shown in green and light green. The putative propofol binding site residues that were mutated to tryptophan at the β “-” surface (Y143, F221, Q224) are colored in yellow, and the residue photolabeled with ortho-propofol diazirine (H267) in orange. The defining propofol site residue mutated at the β “+” surface (M286) is shown in red. Propofol is shown in stick format color-coded by atom type (carbon, black; oxygen red) enclosed in a transparent Connolly surface covering the 20 lowest energy docking solutions for each pocket. Propofol is shown docked in its lowest energy solution at the β-β (A) and β-α (C) interfaces. At the α-β interface (B), the propofol molecule is shown in the lowest energy solution which best agrees with the functional data. The images in the bottom panels were derived from previous docking studies [20].