Permeation properties of inward-rectifier potassium channels and their molecular determinants

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K+ channels ROMK2 (Kir1.1b), IRK 1 (Kir2.1), and their chimeras using heterologous expression in Xenopus ooc ytes. Patch-clamp recordings of single channels were obtained in the cell-a ttached mode with different per-meant cations in the pipette. For inward K conduction, replacing the extracellular loop of ROMK2 with that of IRK1 in creased single-channel conductance by 25 pS (from 39 to 63 pS), whereas rep lacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance b y 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH, terminus or transmembrane domains, suggesting that th e two domains form two resistors in series. The larger conductance of the e xtracellular loop of IRK1 was attributable to a single amino acid differenc e (Thr versus Val) at the 3P position, three residues in front of the GYG m otif. Permeability sequences for the conducted ions were similar for the tw o channels: Tl+ > K+ > Rb+ > NH4+. The ion selectivity sequence for ROMK2 b ased on conductance ratios was NH4+ (1.6) > K+ (1) > Tl+ (0.5) > Rb+ (0.4). For IRK1, the sequence was K+ (1) > Tl+ (0.8) > NH4+ (0.6) >> Rb+ (0.1). T he difference in the NH4+/K+ conductance (1.6) and permeability (0.09) rati os can be explained if NH4+ binds with lower affinity than K+ to sites with in the pore. The relatively low conductances of NH4+ and Rb+ through IRK1 w ere again attributable to the 3P position within the P region. Site-directe d mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred th e relatively high Tl+ conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectiv ity properties of the pore.