M. Jonsson Fagerlund, J. Sjödin, J. Krupp, M.A. Dabrowski

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Reduced effect of propofol at human α1β2(N289M)γ2 and α2β3(N290M)γ2 mutant GABAA receptors† M. Jonsson Fagerlund, J. Sjödin, J. Krupp, M.A. Dabrowski British Journal of Anaesthesia Volume 104, Issue 4, Pages 472-481 (April 2010) DOI: 10.1093/bja/aeq023 Copyright © 2010 The Author(s) Terms and Conditions

Fig 1 Using the SWISSPROT database for protein sequences, TM segment predictions, and numbering, we aligned the second TM region in the β2 and β3 subunits of GABAA receptors from humans (accession no. P47870, Q16323, Q4FZB2) and rats (accession no. P63138, P15432). The sequence alignment is shown together with the full topology and orientation of a β subunit in the cell membrane. The predicted position of the mutations in β2(N289M) and β3(N290M) in TM2 of the human receptor is indicated. British Journal of Anaesthesia 2010 104, 472-481DOI: (10.1093/bja/aeq023) Copyright © 2010 The Author(s) Terms and Conditions

Fig 2 Concentration–response relationship for γ-aminobutyric acid (GABA) at wild-type and mutant human GABAA receptors expressed in Xenopus oocytes. Current responses to a 20 s application of 1, 10, 100, and 1000 µM GABA in wild-type and mutant GABAA receptors. The arrow indicates the start of ion influx into the oocyte. (a) Single currents recorded in each oocyte are superimposed. Responses in each oocyte were normalized to the maximal GABA-evoked peak current in each oocyte, giving the concentration–response curves. (b) Each symbol represents mean (sem) of 8–14 oocytes. When no error bars are seen, they are smaller than the symbols. British Journal of Anaesthesia 2010 104, 472-481DOI: (10.1093/bja/aeq023) Copyright © 2010 The Author(s) Terms and Conditions

Fig 3 Concentration–response curves for propofol at wild-type (wt) (α1β2γ2, α2β2γ2, α2β3γ2) and mutant [α1β2(N289M)γ2, α2β3(N290M)γ2] GABAA receptor subtypes in the presence of 2.5 µM GABA. (a) Data are presented as mean (sem) of 6–13 oocytes. When no error bars are seen, they are smaller than the symbols. Current traces showing a 20 s application of 2.5 µM GABA (left side) and 3 µM propofol (prop)+2.5 µM GABA (right side) in wild-type and mutant receptors (b). British Journal of Anaesthesia 2010 104, 472-481DOI: (10.1093/bja/aeq023) Copyright © 2010 The Author(s) Terms and Conditions

Fig 4 Concentration–response curves for propofol at wild-type (α1β2γ2, α2β2γ2, α2β3γ2) and mutant [α1β2(N289M)γ2, α2β3(N290M)γ2] GABAA receptor subtypes. Current responses to a 20 s application of 10, 30, and 100 µM propofol in wild-type and mutant GABAA receptors (a). Single currents recorded in each oocyte are superimposed. Data are normalized to maximal response to propofol in each oocyte, giving the concentration–response curves. For α1β2(N289M)γ2 and α2β3(N290M)γ2, no curve fit was possible since no plateau was reached at the highest concentration of propofol. (b) Data are presented as mean (sem) of 6–13 oocytes. When no error bars are seen, they are smaller than the symbols. British Journal of Anaesthesia 2010 104, 472-481DOI: (10.1093/bja/aeq023) Copyright © 2010 The Author(s) Terms and Conditions