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 (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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Figure 1: Characterization of ortho-propofol diazirine.
Figure 2: Labeling HSA with ortho-propofol diazirine.
Figure 3: Labeling GABAA receptors with ortho-propofol diazirine.

Change history

  • 27 September 2013

    In the version of this article initially published online, two bars indicating experimental conditions above the current traces in Figure 1a were misplaced. The error has been corrected for the PDF and HTML versions of this article.

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Acknowledgements

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.

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Contributions

C.J.E. and E.H.S. synthesized the propofol photolabels and helped with the data analysis. G.M.S.Y. and Z.-W.C. carried out the labeling experiments, protein chemistry and MS and helped with the analysis. R.D. made and analyzed the electrophysiological measurements. C.J.E. carried out the animal experiments. E.H. contributed to the molecular modeling. W.S. and K.F. supplied membranes containing expressed receptors. R.R.T. contributed to the design and interpretation of MS experiments. A.S.E. and N.P.F. designed and supervised the project, helped with the analysis and wrote the paper.

Corresponding authors

Correspondence to Alex S Evers or Nicholas P Franks.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–6, Supplementary Tables 1 and 2 and Supplementary Note. (PDF 27782 kb)

Supplementary Data Set 1

MS2 spectra of GABAA α1 and β3 subunits (PDF 6552 kb)

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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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