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

Abstract

BackgroundPropofol 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.MethodWe 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.ResultsWe 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.ConclusionWe 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.

GABA Type A Receptor Activation in the Allosteric Coagonist Model Framework: Relationship between EC50 and Basal Activity

“…Figure 4B shows data for receptors containing the a1(L263S) and/or b2(Y143W) mutation. Both mutations have been reported to enhance constitutive activity in the GABA A receptor without altering K GABA (Chang et al, 1996;Chang and Weiss, 1999;Eaton et al, 2016). Introduction of the mutation in either construct shifted the value for L, indicating that both concatemers incorporated in the receptor.…”

“…There is no a priori reason to expect that a mutation that affects receptor activation by the transmitter acts indirectly, solely through changes in basal activity. These two mutations were selected based on prior reports that had indicated minimal effect on K GABA (Chang and Weiss, 1999;Eaton et al, 2016). Mutations that additionally modify K GABA would be expected to exhibit deviations from the predicted, model-based relationship between EC 50 and L. Therefore, compliance with the predicted relationship can serve as indicator of the mechanism of action for a mutation.…”

“…Converting Membrane Current to Probability of Being Open. We adopted the procedures developed by Forman (Forman, 2012;Forman and Stewart, 2012;Eaton et al, 2016) to establish a scale for converting current responses to an estimated probability of being open [P{open}]. The maximal response possible from a given cell (I max ) was estimated by applying a saturating concentration of GABA plus a potentiator that gave the largest measured response.…”

The Actions of Drug Combinations on the GABAAReceptor Manifest as Curvilinear Isoboles of Additivity

“…Photolabeling studies have indicated that there are two classes of sites with two copies of each class of sites per ternary receptor, giving a total of at least four sites (Yip et al, 2013;Jayakar et al, 2014). Previous functional studies have proposed two to five sites per receptor (Ruesch et al, 2012;Eaton et al, 2016;Maldifassi et al, 2016;Nourmahnad et al, 2016;). The data and fits are shown in Fig.…”

Analysis of GABAA Receptor Activation by Combinations of Agonists Acting at the Same or Distinct Binding Sites

“…Data Analysis. The current amplitudes were converted to units of open probability by matching the relative peak responses against a scale ranging from 0 to 1 of the open probability of the receptor (P open ) (Forman and Stewart, 2012;Eaton et al, 2016). Wild-type concatemeric receptors in the absence of agonist exhibit minuscule constitutive activity [i.e., open Fig.…”