Loss of phenomenal external K⁺-free inhibition in Kir2.1 with reduced inward rectification. (A) Whole-cell currents and I–V relationships obtained from the WT channel and point mutants (E224G and E299S) using 5.4 mM K⁺ Tyrode’s (top), Tyrode’s with 5 mM Ba²⁺ (middle), and K⁺-free Tyrode’s (bottom) external solutions. Test pulses were applied from a holding potential of −30 mV, and current amplitudes were measured at the end of the step pulses. Current traces are shown at test potentials between −120 and 0 mV in 10-mV steps. Cells contain endogenous cytoplasmic polyamines. Dashed lines superimposed on current traces indicate the zero-current level. (B) K⁺ efflux through Kir2.1 under the external K⁺-free condition was observed using a polyamine-free, 150-mM K⁺ internal solution. The external solution was switched between 5.4 mM K⁺ (top) and K⁺-free (bottom) Tyrode’s solutions; currents were recorded from an outside-out patch membrane in the sequence indicated alphabetically in the figure (a–e). A depolarizing ramp pulse (1 V s⁻¹) was used to obtain I-V relationships.