Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA (γ-aminobutyric acid type A) receptors, but where it binds to this receptor is not known and has been a matter of some debate. We synthesized a new propofol analog photolabeling reagent whose biological activity is very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin that have been identified using X-ray crystallography. Using a combination of protiated and deuterated versions of the reagent to label mammalian receptors in intact membranes, we identified a new 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 the transmembrane segments TM1 and TM2.
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Hales, T.G. & Lambert, J.J. The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurones. Br. J. Pharmacol. 104, 619–628 (1991).
Jurd, R. et al. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABAA receptor β3 subunit. FASEB J. 17, 250–252 (2003).
Zezula, J., Slany, A. & Sieghart, W. Interaction of allosteric ligands with GABAA receptors containing one, two, or three different subunits. Eur. J. Pharmacol. 301, 207–214 (1996).
Davies, P.A., Kirkness, E.F. & Hales, T.G. Modulation by general anaesthetics of rat GABAA receptors comprised of α1 β3 and β3 subunits expressed in human embryonic kidney 293 cells. Br. J. Pharmacol. 120, 899–909 (1997).
Feng, H.J. & Macdonald, R.L. Multiple actions of propofol on αβγ and αβδ GABAA receptors. Mol. Pharmacol. 66, 1517–1524 (2004).
Jones, M.V., Harrison, N.L., Pritchett, D.B. & Hales, T.G. Modulation of the GABAA receptor by propofol is independent of the γ subunit. J. Pharmacol. Exp. Ther. 274, 962–968 (1995).
Krasowski, M.D. et al. α subunit isoform influences GABAA receptor modulation by propofol. Neuropharmacology 36, 941–949 (1997).
Lam, D.W. & Reynolds, J.N. Modulatory and direct effects of propofol on recombinant GABAA receptors expressed in Xenopus oocytes: influence of α- and γ2-subunits. Brain Res. 784, 179–187 (1998).
Franks, N.P. General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal. Nat. Rev. Neurosci. 9, 370–386 (2008).
Moraga-Cid, G., Yevenes, G.E., Schmalzing, G., Peoples, R.W. & Aguayo, L.G. A single phenylalanine residue in the main intracellular loop of α1 γ-aminobutyric acid type A and glycine receptors influences their sensitivity to propofol. Anesthesiology 115, 464–473 (2011).
O'Shea, S.M., Williams, C.A. & Jenkins, A. Inverse effects on gating and modulation caused by a mutation in the M2–M3 linker of the GABAA receptor γ subunit. Mol. Pharmacol. 76, 641–651 (2009).
Krasowski, M.D. et al. Propofol and other intravenous anesthetics have sites of action on the γ-aminobutyric acid type A receptor distinct from that for isoflurane. Mol. Pharmacol. 53, 530–538 (1998).
Krasowski, M.D., Nishikawa, K., Nikolaeva, N., Lin, A. & Harrison, N.L. Methionine 286 in transmembrane domain 3 of the GABAA receptor β subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. Neuropharmacology 41, 952–964 (2001).
Chang, C.S., Olcese, R. & Olsen, R.W. A single M1 residue in the β2 subunit alters channel gating of GABAA receptor in anesthetic modulation and direct activation. J. Biol. Chem. 278, 42821–42828 (2003).
Siegwart, R., Krahenbuhl, K., Lambert, S. & Rudolph, U. Mutational analysis of molecular requirements for the actions of general anaesthetics at the γ-aminobutyric acid A receptor subtype, α1β2γ2. BMC Pharmacol. 3, 13 (2003).
Richardson, J.E. et al. A conserved tyrosine in the β2 subunit M4 segment is a determinant of γ-aminobutyric acid type A receptor sensitivity to propofol. Anesthesiology 107, 412–418 (2007).
Williams, C.A., Bell, S.V. & Jenkins, A. A residue in loop 9 of the β2-subunit stabilizes the closed state of the GABAA receptor. J. Biol. Chem. 285, 7281–7287 (2010).
Siegwart, R., Jurd, R. & Rudolph, U. Molecular determinants for the action of general anesthetics at recombinant α(2)β(3)γ(2)γ-aminobutyric acid(A) receptors. J. Neurochem. 80, 140–148 (2002).
Nury, H. et al. X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel. Nature 469, 428–431 (2011).
Bali, M. & Akabas, M.H. Defining the propofol binding site location on the GABAA receptor. Mol. Pharmacol. 65, 68–76 (2004).
Li, G.D., Chiara, D.C., Cohen, J.B. & Olsen, R.W. Numerous classes of general anesthetics inhibit etomidate binding to γ-aminobutyric acid type A (GABAA) receptors. J. Biol. Chem. 285, 8615–8620 (2010).
Chen, Z.W. et al. Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABAA receptor. Mol. Pharmacol. 82, 408–419 (2012).
Chen, Z.W., Fuchs, K., Sieghart, W., Townsend, R.R. & Evers, A.S. Deep amino acid sequencing of native brain GABAA receptors using high-resolution mass spectrometry. Mol. Cell. Proteomics 11, M111.011445 (2012).
Waud, D.R. On biological assays involving quantal responses. J. Pharmacol. Exp. Ther. 183, 577–607 (1972).
Bhattacharya, A.A., Curry, S. & Franks, N.P. Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures. J. Biol. Chem. 275, 38731–38738 (2000).
Bright, D.P. et al. Identification of anesthetic binding sites on human serum albumin using a novel etomidate photolabel. J. Biol. Chem. 282, 12038–12047 (2007).
Kang, S.U., Fuchs, K., Sieghart, W. & Lubec, G. Gel-based mass spectrometric analysis of recombinant GABAA receptor subunits representing strongly hydrophobic transmembrane proteins. J. Proteome Res. 7, 3498–3506 (2008).
Hibbs, R.E. & Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011).
Wooltorton, J.R., McDonald, B.J., Moss, S.J. & Smart, T.G. Identification of a Zn2+ binding site on the murine GABAA receptor complex: dependence on the second transmembrane domain of β subunits. J. Physiol. (Lond.) 505, 633–640 (1997).
James, R. & Glen, J.B. Synthesis, biological evaluation, and preliminary structure-activity considerations of a series of alkylphenols as intravenous anesthetic agents. J. Med. Chem. 23, 1350–1357 (1980).
Cestari, I.N., Min, K.T., Kulli, J.C. & Yang, J. Identification of an amino acid defining the distinct properties of murine β1 and β3 subunit-containing GABAA receptors. J. Neurochem. 74, 827–838 (2000).
Carlson, B.X., Engblom, A.C., Kristiansen, U., Schousboe, A. & Olsen, R.W. A single glycine residue at the entrance to the first membrane-spanning domain of the γ-aminobutyric acid type A receptor β(2) subunit affects allosteric sensitivity to GABA and anesthetics. Mol. Pharmacol. 57, 474–484 (2000).
Li, G.D. et al. Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J. Neurosci. 26, 11599–11605 (2006).
Hall, M.A. et al. m-Azipropofol (AziPm) a photoactive analogue of the intravenous general anesthetic propofol. J. Med. Chem. 53, 5667–5675 (2010).
Stewart, D.S. et al. p-(4-Azipentyl)propofol: a potent photoreactive general anesthetic derivative of propofol. J. Med. Chem. 54, 8124–8135 (2011).
Evers, A.S. et al. A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors. J. Pharmacol. Exp. Ther. 333, 404–413 (2010).
Darbandi-Tonkabon, R. et al. Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain. J. Biol. Chem. 278, 13196–13206 (2003).
Nesvizhskii, A.I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 (2003).
We thank the Medical Research Council (UK) (grant G0901892 to N.P.F.), the US National Institutes of Health (NIH) - National Institute of General Medical Sciences (grant PO1-GM47969 to A.S.E. and grant 8 P41 GM103422-35 to R.R.T.), the Austrian Ministry of Science and Research and the European Seventh Framework Program (grant HEALTH-F4-2008-202088 to W.S.) for support. We also thank R. Yustos for technical assistance and D. Droste, M. Bennett, T. Gent, K. Karu, B. Manion, R. Ruby and J. Malone for help with the experiments.
The authors declare no competing financial interests.
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Yip, G., Chen, Z., Edge, C. et al. A propofol binding site on mammalian GABAA receptors identified by photolabeling. Nat Chem Biol 9, 715–720 (2013). https://doi.org/10.1038/nchembio.1340
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