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

Figure 3. Functional assessment of mTREK1 residues identified by azi-isoflurane photolabeling.

Representative two electrode voltage clamp recordings of mTREK1 wildtype (WT) and channel mutants G182W and D194W. (A) Basal current measured 24 hr after microinjection of 2.4 ng cRNA. (B) Temperature dependence of TREK1 currents, measured at temperatures of 20–35°C, in 5°C increments. (C) Response to administration of 2 mM isoflurane, followed by washout. (D) Response to administration of 10 μM BL1249. For temperature, isoflurane, and BL1249, experiments performed on TREK1 channels bearing mutations that alter basal current density, the concentration of microinjected cRNA was titrated to achieve 1 μA of current at 20°C, to approximate WT channel current density. (E) Quantification of TREK1 channel activity on basal current level, (F) temperature dependence as measured by Q10 (30°C/20°C), (G) response to isoflurane administration, (H) changes in external pH, or (I) BL1249 administration, as measured by TREK1 current at 0 mV. Number of replicate experiments indicated. Statistical significance was determined by one-way ANOVA combined with a Dunnetts multiple comparison test against mTREK1 WT data, results indicated, *p<0.5, **p<0.05, ****p<0.0005. Error bars are mean ± SEM.

Figure 3.

Figure 3—figure supplement 1. Function assessment of the drTREK1 isoform.

Figure 3—figure supplement 1.

(A) Pairwise alignment of the mouse and zebrafish TREK1 protein sequences, with the transmembrane domains (TM), extracellular cap (CAP), and selectivity filter (SF) sequences denoted. (B) Representative traces showing temperature dependence of drTREK1 current, measured at temperatures of 20–35°C, in 5°C increments and (C) quantification of temperature responsiveness as measured by drTREK1 current at 0 mV. (D) Representative traces of drTREK1 response to administration of 2 mM Isoflurane or (E) 10 μM BL1249. Experiments performed on mTREK1 channels bearing mutations that alter basal current density, the concentration of microinjected cRNA was titrated to achieve 1 µA of current at 20°C, to approximate wild-type channel current density. (E) Quantification of mTREK1 channel activity on basal current level, (F) temperature dependence as measured by Q10 (30°C/20°C), (G) response to isoflurane administration, (H) changes in external pH, or (I) BL1249 administration, as measured by mTREK1 current at 0 mV. Number of replicate experiments indicated. Error bars are mean ± SEM. Statistically significance was determined in panels F–H by unpaired two tailed t-tests. Results indicated, **p<0.05, ****p<0.0005.