A propofol binding site on mammalian GABAA receptors identified by photolabeling

Abstract

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2.

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

“…interface of a pocket accessible from the ion channel and in contact with ␤3His-267, the residue photolabeled by o-PD (20). Also included in the figure is an alignment of ␣1 and ␤3 subunit sequences in the M1-M3 transmembrane domain with the photolabeled amino acids highlighted.…”

“…10 with AziPm docked in the lowest energy orientation predicted by computational docking and the amino acids identified in ␣M1, ␤M1, and ␤M3 that are photolabeled by [ 3 H]AziPm and in ␤M2 by o-PD (␤3His-267 (␤M2-17Ј) (20)). Also highlighted are the amino acids photolabeled by photoreactive etomidates and R-mTFD-MPAB and those identified by mutational analyses as propofol sensitivity determinants (␤3M2-15Ј (␤3Asn-265), ␤3M3-4Ј (␤3Met-286), and ␤3Tyr-442 in ␤M4 (7-9)).…”

“…o-PD was synthesized from 2-isopropyl-6-trifluoroacetylphenol as described (20) with the purity Ͼ96% as judged by 1 H and 19 F NMR. As reported (20), the UV spectrum was characterized by an absorption maximum at 280 nm (extinction coefficient, ⑀ 280 ϭ 2417 Ϯ 24 M Ϫ1 cm Ϫ1 ) and an unresolved long wavelength shoulder (⑀ 350 ϭ 66 Ϯ 1 M Ϫ1 cm…”

“…Although etomidate bound selectively to the ␤ ϩ interface sites and certain barbiturates bound selectively at the ␤ Ϫ interface sites, propofol inhibited photolabeling at both classes of sites, but only at concentrations (IC 50 ϳ40 M) that were ϳ10-fold higher than the concentrations necessary to potentiate GABA responses. This discrepancy suggests that propofol may bind with higher affinity to other, as yet unidentified, sites in the GABA A R. A reactive propofol analog (o-propofol diazirine (o-PD)) was recently shown to photoincorporate in expressed ␣1␤3 GABA A R into ␤3His-267 (␤M2-17Ј), an amino acid in the ␤ subunit M2 helix in proximity to the R-mTFD-MPAB site, but projecting into the lumen of the ion channel near the interface between the extracellular and transmembrane domains (20).…”

“…10, E and F). ␤3His-267, photolabeled by o-PD (20), contributes to the channel lumen and the interface between M2 helices, one helical turn above and ϳ11 Å from ␣1Ser-270 (␣M2-15Ј) or ␤3Asn-265 (␤M2-15Ј) and ϳ15 Å from ␤3Met-227. As noted in the structure of the ␤3 GABA A R (11), ␤3His-267 contributes to a potential propofol binding pocket that is accessible from the lumen of the ion channel.…”