Extracellular D₂O does not alter the midpoint of the voltage dependence of steady-state activation of Shaker-IR mutant channels expressed in tsA201 cells. (A–C) Cells were held at −120 mV and depolarized to gradually increasing test potentials ranging from −100 to +70 mV in steps of 10 mV in every 45 s. The duration of the depolarizing pulses was 150 ms. G–V curves were constructed from current–voltage relationships using G(V) = I_peak/(V_test − E_K) with the following parameters: peak current (I_peak) at a test potential of V_test and a K⁺ reversal potential of E_K = −85 mV. The conductance values were normalized to the maximum (G/G₀) and plotted as a function of the test potential (E_m). The voltage dependencies of steady-state activation curves were determined in the absence (filled symbols, H₂O//H₂O condition) or presence of extracellular D₂O (empty symbols, H₂O//D₂O condition). The superimposed solid lines show the best-fit Boltzmann functions to the averaged data points. (D) Bars and error bars indicate the mean ± SEM (n ≥ 4) of the midpoint voltages (V_1/2) of steady-state activation 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).