[K⁺]_out dependence of the polyamine-unblocked Kir2.1 conductance. (A) Representative currents recorded from an outside-out patch membrane at different [K⁺]_out levels (150, 50, and 20 mM) in the absence (top) and presence (bottom) of Na⁺ substitution ([K⁺]_out + [Na⁺]_out = 150 mM). Holding potentials were set near the theoretical E_K value at each [K⁺]_out, and a 10-ms hyperpolarizing prepulse (60 mV negative to HP) ensured relief of any polyamine block before applying test pulses. Traces are at potentials between −80 and 80 mV relative to the holding potentials. Dashed lines superimposed on current traces indicate the zero-current level. (B) Instantaneous I–V relationships at the onset of test pulses obtained from the currents shown in A. (C) Chord conductances (G) plotted against V–V_rev, obtained from the I–Vs shown in B. (D) Voltage dependence of Na⁺ inhibition. Fractional inhibition of conductances observed with Na⁺ substitution at 50 mM and 20 mM [K⁺]_out was obtained from the data shown in C and was plotted against V. Lines are the fits to the Boltzmann equation: fractional inhibition = 1/{1 + exp[−(V − V_1/2)]/s}, with s = 86 mV. In this experiment, the electrical distance (δ) of the Na⁺ blocking site is ∼0.3 from the outside. (E) [K⁺]_out dependence of the polyamine-unblocked conductance with and without Na⁺ substitution. Relative G values (normalized to the value at 150 mM [K⁺]_out (0 Na⁺) in each experiment) are plotted against external K⁺ activity on double-logarithmic coordinates (left: V_rev −40 mV; right: V_rev +40 mV). Error bars represent SD. The slopes of the dashed lines are 0.29 and 0.22 in the left and right panels, respectively.