Fig. 4. The effects of combining S2 point mutations and S4 insertional mutations on steady-state activation properties. In all panels the wild-type jShak1 channel is indicated by the red circles, the S2 single mutants are indicated by triangles, the S4 insertion mutants are indicated by squares and the double mutants are indicated by diamonds. (A,C,E) The effect of combining the N227D mutation with the S4 triplet insertions in the double mutant channels. (B,D,F) The effect of combining the N227E mutation with the S4 triplet insertions in the double mutant channels. The solid curve represents the fit to a Boltzmann function. Voltage of half activation (V₅₀) and Boltzmann slope factors (b) for each channel are summarized in Table 1 (error bars indicate s.e.m.). (A)The effect of the N227D mutation in the S2 helix in combination with the QIF insertion mutation in S4 is similar to its effect on the wild-type channel. There appears to be a slight shift in slope factor and V₅₀, but the effect is not statistically significant. (B)The steady-state activation curve for the QIF-E double mutant (pink diamonds), the single QIF mutant (green squares), and the N227E point mutant (black triangles) were all shifted leftward approximately the same amount relative to the wild-type channel (red circles), but had different slopes, although the differences were not statistically significant. (C)The steady-state activation curves for the IFR-D double mutant (turquoise diamonds), the single mutant N227D (orange triangles), IFR insertion alone (green squares) were not significantly different from the wild-type channel (red circles). (D)The steady-state activation curve for the IFR-E double mutant (pink diamonds) was shifted in a hyperpolarized direction relative to the IFR mutant alone (green squares) and the wild-type channel (red circles) and had a similar V₅₀ of activation to that of the N227E single mutant (blue triangles), but with a larger slope factor, b. (E)The steady-state activation curve for the RIF-D double mutant (turquoise diamonds) was shifted in a depolarized direction relative to the single N227D mutant (orange triangles), to the same extent that the RIF mutant (green squares) was shifted relative to the wild-type channel (red circles). (F)The steady-state activation curve for the RIF-E double mutant (pink diamonds) was shifted in a depolarized direction relative to the wild-type channel (red circles), which was in the opposite direction to the shift for the N227E single mutant (black triangles). The RIF-E double mutant is also shifted in the hyperpolarizing direction relative to the RIF mutant alone (green squares).