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. 2021 May 20;153(6):e202012742. doi: 10.1085/jgp.202012742

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© 2021 Szanto et al.

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Figure 6. — Effect of extracellular D₂O substitution on the kinetics of recovery from inactivation at −120 mV. (A–C) The time course of recovery from inactivation of the K⁺ current of Shaker-IR mutants was studied by means of a two-pulse protocol with increasing interpulse time (ipi from 0.5 to 60 s; see Materials and methods for details) between the pulses for T449A (A), T449A/I470A (B), and T449K/I470C (C). Data points 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). The time constants of recovery (τ_rec) were determined by fitting the averaged data points with a single-exponential function (see Materials and methods); the superimposed solid lines show the best fits in all panels. (D) Bars and error bars indicate the mean ± SEM (n ≥ 4) of the τ_rec values 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) and H₂O//D₂O (empty symbols).