Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Abstract

Background: Propofol binding to GABA A R sites of uncertain location potentiates receptor function and produces anesthesia in vivo. Results: A photoreactive propofol analog identifies propofol-binding sites in ␣1␤3 GABA A Rs. Conclusion: Propofol binds to each class of intersubunit sites in the GABA A R transmembrane domain. Significance: This study demonstrates that propofol binds to the same sites in a GABA A R as etomidate and barbiturates.

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β− Interface

“…Although etomidate and R-mTFD-MPAB bind with Ͼ50-fold selectivity to the ␤ ϩ -or ␤ Ϫ -containing interface sites, respectively, the sites are not strictly etomidate-or barbiturate-specific. Propofol binds with little selectivity to both classes of sites, and a propofol analog photolabels residues in both classes of sites (23,24). These allosteric anesthetic-binding sites in the TMD show positive energetic coupling to each other and to the GABA-binding sites at the ␤ ϩ -␣ Ϫ subunit interfaces in the extracellular domain (ECD), as etomidate and GABA each enhanced R- [ 3 H]mTFD-MPAB photolabeling.…”

“…Our results provide a first demonstration that, similar to the benzodiazepine-binding site at the ␣ ϩ -␥ Ϫ interface in the ECD (27), at least one intersubunit-binding site in the GABA A R's TMD is a target for negative as well as positive allosteric modulators. (24). Similar to mTFD-MPAB, nonradioactive (Ϯ)-mTFD-MPPB (5-propyl-1-methyl-5-(m-trifluoromethyldiazirynylphenyl)barbituric acid) was synthesized by reaction of 5-propyl-1-methyl barbiturate with (4-methoxyphenyl)-[3-(3-trifluoromethyl-3H-diazirin-3-yl) phenyl]iodonium trifluoroacetate, and preparative separation of R-and S-mTFD-MPPB was performed by chiral chromatography on a Chiralpak IC column.…”

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

“…However, previous work has demonstrated that propofol (and other anesthetic drugs) may act via the b(1)-a(2) interface (Krasowski et al, 2001a;Bali and Akabas, 2004;Li et al, 2006;Jayakar et al, 2014), that remained unmodified in a1(I271W)b3 receptors. To additionally introduce the tryptophan substitution to the b-a interface, we combined a1(I271W) and b3(T266W) subunits.…”

“…The a1b3 receptor thus has one b-b, two b-a, and two a-b interfaces. Mutations to the b3(H267) residue can be expected to modify and affect the b-b and a-b interfaces, but not the b-a interface, whose involvement in the actions of propofol has been suggested by previous photolabeling and functional studies (Krasowski et al, 2001a;Bali and Akabas, 2004;Jayakar et al, 2014). It is therefore plausible that the contribution from the unaltered b-a interfaces conceals the true effect exerted by the H267A mutation at the "2" side of the b subunit.…”

“…If we assume that mutations to the "2" side of b3 affect both b-b and a-b type interfaces, that still leaves two unmodified b-a interfaces. A recent study that employed the photoreactive propofol analog 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol demonstrated labeling at the b-a interface in human a1b3 receptors (Jayakar et al, 2014). Interestingly, the residues labeled [b3(M286), a1(M236), and a1(I239)] are located in a plane more cytoplasmic relative to the key residues in b3 homomers in the long axis of membranespanning domains.…”