Effect of R475 and R479 mutations on the PIP₂ modulation of Elk1. (A and B) R475E and R479E current traces recorded in response to 1-s voltage steps (−120 to 60 mV in 20-mV increments; −100-mV holding potential) in the on-cell configuration. (Insets) Normalized current traces at 20 mV for R475E and at −20 mV for R479E (reversed in polarity for display) comparing on cell (black), excised (red), and plus 10 µM PIP₂ (blue). (C) τ_act(10 µM PIP₂)/τ_act(Ctrl) at various voltages compared for R475E (n = 20), R479E (n = 10), and WT (n = 18). R479E differed from WT as indicated: *, P < 0.05, or ***, P < 0.001. (D) G-V mode shift determined with protocols shown in Fig. 2 A for the indicated mutants and conditions (n numbers in Table 2; ***, P < 0.001; t test). (E and F) G-V curves for R475E and R479E on cell (black), excised (red), or plus 10 µM PIP₂ (blue; dotted lines, corresponding WT). V₅₀, s, and n numbers are reported in Table 1. (G–I) Repeat of C–E for R479Q. R479Q, n = 5 in G; H and I, sample numbers are reported in Tables 1 and 2, with ΔV₅₀ and Boltzmann parameters, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. WT; t test). All data are mean ± SEM, and curves in E, F, and I show single Boltzmann fits.