Fig. 3. Single mutations in the S2 helix and triplet insertions in the S4 helix modify steady-state activation properties. (A)The steady-state activation curve for jShak1 S2 mutants revealed that the N227E (inverted triangles) channels were activated at far more hyperpolarized potentials than wild-type channels (filled circles) and N227D mutant channels (open circles). The solid curve represents the fit to a Boltzmann function giving the voltage of half activation (V₅₀) and slope parameter (b) for each channel (error bars indicate s.e.m.). Fit parameters are summarized in Table 1. (B)The steady-state activation curve for jShak1 S4 insertion mutants revealed that insertion of the QIF motif (inverted triangles) to the N-terminal side of K294 shifted the activation curve in the hyperpolarized direction, the RIF insertion (open circles) shifted the activation curve in the depolarized direction, and the IFR insertion (open triangles) had no significant effect compared with wild-type channels (filled circles). 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.).