G protein–coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gβγ subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gβγ. Mutations in TM2 were found that mimicked the Gβγ-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gβγ subunits but depended on PtdIns(4,5)P₂. The results suggest that the TM2 region plays a key role in channel gating following Gβγ binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K⁺ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.