Inwardly rectifying potassium channels (K(ir)), comprising four subunits each with two transmembrane domains, M1 and M2, regulate many important physiological processes. We employed a yeast genetic screen to identify functional channels from libraries of K(ir) 2.1 containing mutagenized M1 or M2 domains. Patterns in the allowed sequences indicate that M1 and M2 are helices. Protein-lipid and protein-water interaction surfaces identified by the patterns were verified by sequence minimization experiments. Second-site suppressor analyses of helix packing indicate that the M2 pore-lining inner helices are surrounded by the M1 lipid-facing outer helices, arranged such that the M1 helices participate in subunit-subunit interactions. This arrangement is distinctly different from the structure of a bacterial potassium channel with the same topology and identifies helix-packing residues as hallmark sequences common to all K(ir) superfamily members.