Current–voltage relationships for T449A/I470A Shaker-IR mutant channels at various [K⁺] combinations in inside-out patches. (A) The inside-out patch was held at a holding potential of −120 mV and depolarized to +50 mV for 5 ms to fully activate the channels (not shown). This step is followed by a fast voltage ramp to −120 mV in 25 ms. The currents corresponding to the membrane potential range of +50 mV to −60 mV are shown for clarity. The extracellular solution contained 50 mM K⁺ prepared in H₂O (H₂O//H₂O). The patch was perfused with an intracellular solution containing 150 mM K⁺ (red trace) or 15 mM K⁺ (green trace). The horizontal dotted line indicates the 0-pA current, and the vertical dashed lane drawn at 0 mV indicates that the current is inward at [K⁺]_in//[K⁺]_ex = 15//50 mM (green trace) and outward at [K⁺]_in//[K⁺]_ex = 150//50 mM (red trace). (B) The same set of experiments as in A, except that the pipette-filling extracellular solution containing 50 mM K⁺ was prepared in D₂O (H₂O//D₂O). All others (e.g., [K⁺] and labels) are the same as in A. (C) Current reversal potential (V_R) was determined from the traces shown in A and B as the membrane potential at which the current crosses the 0-pA reference line (dotted). Bars and error bars indicate the mean ± SEM (n = 5–6) of the V_R values, and symbols (filled and empty circles) indicate individual data points obtained in H₂O//H₂O solution (filled symbols) and extracellular D₂O (H₂O//D₂O, empty symbols).