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.

Many GABAergic drugs are in clinical use as anesthetics, sedatives, or anxiolytics. We have investigated the actions of the combinations of the neuroactive steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with the intravenous anesthetic propofol or the benzodiazepine diazepam. The goal of the study was to determine whether coapplication of alfaxalone reduces the effective doses and concentrations of propofol and diazepam. Behavioral effects of alfaxalone, propofol, diazepam, and the combinations of the drugs were evaluated during a 30-min activity test in mice. Functional effects of the individual drugs and drug combinations were tested by measuring the decay times of spontaneous inhibitory postsynaptic currents in rat hippocampal neurons, and peak current responses from heterologously expressed concatemeric α1β2γ2L GABAA receptors. Co-administration of alfaxalone increased the sedative actions of propofol and diazepam in mice. The combination of alfaxalone with propofol or diazepam increased the decay times of sIPSCs and shifted the concentration-response relationships for GABA-activated receptors to lower transmitter concentrations. We infer that alfaxalone acts as a co-agonist to enhance the GABAergic effects of propofol and diazepam. We propose that co-administration of alfaxalone, and possibly other neuroactive steroids, can be employed to reduce dosage requirements for propofol and diazepam.

Propofol is a sedative and anesthetic agent that can both activate GABA A receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the b3 homomeric GABA A receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human b3 and a1b3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the b3(Y143), b3(F221), b3(Q224), and b3(T266) residues in the actions of propofol but not pentobarbital in b3 receptors. The effects of b3(Y143W) and b3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in b3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the a1 subunit had almost no effect on activation properties in a1b3 heteromeric receptors. We hypothesize that heteromeric a1b3 receptors can be activated by propofol interactions with b3-b3, a1-b3, and b3-a1 interfaces, but the exact locations of the binding site and/ or nature of interactions vary in different classes of interfaces.