Analysis of the recovery from inactivation kinetics using the sum of two exponentials terms. (A–C) The time course of recovery from inactivation of the K⁺ current of Shaker-IR constructs was studied by means of a double-pulse protocol with increasing interpulse time (ipi; see text for details) for T449A (A), T449A/I470A (B), and T449K/I470C (C). Data points are presented as FR = (I₂ − I_SS1) / (I₁ − I_SS1), where I₂ and I₁ are the peak currents during the second and the first pulse and I_SS1 is the steady-state current at the end of the first depolarization, respectively, and plotted as a function of ipi in control conditions (H₂O//H₂O, filled symbols) and external D₂O (H₂O//D₂O, open symbols). Error bars indicate the SEM. The time constants of recovery were determined by fitting the averaged data points with a sum of two exponential terms, and solid lines show the best fit. (D and E) Time constants characterizing the fast (τ_fast; D) and slow components (τ_slow; E) of the recovery from inactivation were determined for each cell in control (H₂O//H₂O, filled symbols) and heavy water–based extracellular solution (H₂O//D₂O, empty symbols); the mean ± SEM and individual data points are shown for the indicated constructs (circles, T449A; down triangles, T449A/I470A; up triangles, T449K/I470C). The small number of data points for the τ_fast and τ_slow in case of the T449A/I470A and T449K/I470C mutants indicate the failure of the fit with a sum of two exponential terms; the recovery kinetics was basically single exponential for these mutants (Fig. 6).