Effect of extracellular D₂O substitution on the voltage dependence of steady-state inactivation. (A–C) Steady-state inactivation of Shaker-IR mutants was determined by depolarizing inside-out patches at +50 mV for 5 ms from a holding potential of −120 mV to elicit potassium currents (I₋₁₂₀) and then applying a series of 3-s conditioning prepulse potentials ranging from −120 mV to −30 mV in 10-mV increments, with each voltage step followed by a constant 5-ms test pulse to +50 mV to determine I. Potassium currents recorded at each prepulse potential were normalized to their respective maximal values (I/I₋₁₂₀), averaged, and plotted as a function of the prepulse potential in control conditions (H₂O//H₂O, filled symbols) and extracellular D₂O (H₂O//D₂O, empty symbols) for T449A (A), T449A/I470A (B), and T449K/I470C (C), respectively. (D) Bars and error bars indicate the mean ± SEM (n ≥ 3) of the midpoint voltages (V_1/2) of the voltage dependence of steady-state inactivation obtained for the indicated clones. Symbols indicate individual data points (circles, T449A; down triangles, T449A/I470A; up triangles, T449K/I470C) obtained in H₂O//H₂O (filled symbols) as control and H₂O//D₂O (empty symbols). Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01).