Extended Data Fig. 5: Mechanisms of halothane inhibition and lipid activation of THIK-1.
a,b, Analysis of halothane inhibition for WT and THIK-1 R87D mutant channels, respectively showing that the R87D channel retains similar sensitivity to WT THIK-1. c, Excised patch recordings of 1 s duration at −100 mV of single R87D THIK-1 channels in the absence (control) and presence of 0.4 % (upper panel) or 1% halothane (lower panel). The dotted lines represent zero current levels. Halothane induces destabilisation (shortening) of both open and closed states. Thus, because destabilisation of channel closings increases Po, then destabilisation of channel openings must drive the inhibitory effect of halothane. Consistent with this, higher concentrations of halothane (1% lower panel) induced a further, more dramatic reduction in the duration of openings, resulting in ‘flickery’ single-channel kinetics and an even greater decrease in channel Po (30% and 75% decrease in activity in the presence of 0.4% and 1% halothane, respectively. d, Representative recording from an inside out patch containing WT THIK-1 with symmetrical K+ concentrations (120 mM [K+]) at pH 7.4. Channel currents were activated with indicated concentrations of linoleic acid. e, Analysis of fold activations from recordings as in panel f for WT R92A and F262A mutants showing reduced effect on linoleic acid activation but no effect on oleoyl-CoA activation. All values shown in this figure are given as mean ± s.e.m with number (n) of individual recordings indicated above the bars or in the legend. f, EC50 values from dose-response curves as in panel e for WT and mutant THIK-1. g, Fold activation of WT and R92A and F262A mutant THIK-1 channels, respectively with either 30 µM linoleic acid or 5 µM oleoyl-CoA.
