Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human β3 Homomeric GABA_A Receptors

Abstract

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.

Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes

“…Mutations at photolabeled inter-subunit sites alter both receptor function and anesthetic sensitivity, supporting the pharmacological relevance of these sites (56–58). Molecular modeling studies also suggest that anesthetic binding to inter-subunit pockets in GABA A Rs correlates with drug potency (59).…”

Sites of Anesthetic Inhibitory Action on a Cationic Ligand-Gated Ion Channel

“…Affinity-labeling studies of the GABA A R provided more detail on this IS cavity (Jayakar et al, 2014;Yip et al, 2013), corroborated by the crystal structure of the receptor presenting a large cavity precisely at the IS location (Miller and Aricescu, 2014). A structural interpretation on propofol binding followed (Franks, 2015), substantiated by a mutational study (Eaton et al, 2015). The recent cryo-microscopy structures of zebrafish GlyR show that ivermectin may come close to this cavity as well (Du et al, 2015).…”

A Cysteine Substitution Probes β3H267 Interactions with Propofol and Other Potent Anesthetics in α1β3γ2L γ-Aminobutyric Acid Type A Receptors

“…However, our “positive control” finding that mTFD-MPAB protects β3H267C indicates that this is unlikely and that the technique worked as intended. Moreover, a recent study of β3H267W effects in β3 and α1β3 also found no evidence for propofol interactions with this residue 41 .…”