IRK1 currents were recorded from inside-out membrane patches of Xenopus oocytes (injected with IRK1 cRNA) with an Axopatch 200B amplifier (Axon Instruments, Inc.), filtered at 5 kHz, and sampled at 25 kHz using an analogue-to-digital converter (DigiData 1200; Axon Instruments, Inc.) interfaced with a personal computer. pClamp6 software (Axon Instruments, Inc.) was used to control the amplifier and acquire the data. During current recording, the voltage across the membrane patch was first hyperpolarized from the 0 mV holding potential to −100 mV and then stepped to various test voltages, or stepped directly from the holding potential. The duration of the voltage test pulse was 100 ms, which is comparable to those used in the studies where the Mg2+- and polyamine-independent rectification was initially observed (Aleksandrov et al., 1996 (link); Shieh et al., 1996 (link)). Background leak current correction was performed as described previously (Lu and MacKinnon, 1994 (link); Guo and Lu, 2000a (link)). To effectively perfuse the patch, the tip of the patch pipette (∼3 MΩ) was immersed in a stream of intracellular solution exiting one of ten glass capillaries (ID = 0.2 mm) mounted in parallel.