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. 2020 Dec 21;9:e59839. doi: 10.7554/eLife.59839

Figure 7. Mutations in the isoflurane-binding site alter anesthetic sensitivity without perturbing global channel function.

(A) Representative MD) snapshot showing the isoflurane-binding site, including residues predicted to have >20% occupancy by isoflurane shown in sphere representation and isoflurane shown in stick representation (lime). (B) Alignments of mouse TREK1 and TRAAK sequences, with the isoflurane-binding domain regions of TREK1 TM2, TM3, and TM4 (as identified by MD simulation) highlighted in blue. Arrows denote positions of high isoflurane occupancy. Poorly conserved residues are color coded throughout the figure [F185 (blue), G186 (green), T211 (red), and M291 (black)]. (C) Quantification of TREK1 wildtype and mutant responses to 2 mM isoflurane administration, (D) temperature, as measured by TREK1 current at 0 mV, or (E) temperature dependence as measured by Q10 (30°C/20°C). Number of replicate experiments indicated. Error bars are mean ± SEM. Statistically significance was determined by one-way ANOVA combined with a Dunnetts multiple comparison test against mTREK1 WT data, results indicated, **p<0.05,****p<0.0005.

Figure 7.

Figure 7—figure supplement 1. Mass spectrometry (MS) analysis of purified human TRAAK.

Figure 7—figure supplement 1.

Results of MS analysis of TRAAK in the absence of reaction with azi-isoflurane (top) or following reaction with 30 μM azi-isoflurane (bottom). Regions positively identified by MS analysis are shown in blue in the TRAAK structural model (PDB ID 4WFE) and in black font in the sequence data. Regions absent from MS data are displayed in matching color in both the structural model and the sequence data. TRAAK residues homologous to TREK1 positions that exhibit high isoflurane occupancy in MD simulation are displayed as spheres in the structural model of TRAAK and are denoted in the sequence data by an enlarged font. All these residues are identified by MS, but none show evidence of azi-isoflurane labeling. A group of residues in the C-terminal region of TRAAK (marked in gray in the sequence data) were absent from our MS analysis but are not present in the TRAAK structural model.