Strategy: identify pairs of sites that show charge–charge compensation. (A, left) Topology of K_IR2.1 indicating the P-loop location where lysine suppressed current (T141K; yellow circle) and a site in TM2 where second site mutation to aspartate restored conduction (green circle; Chatelain et al., 2005). (right) Predicted topology of K_2PØ indicating two domains (I and II), two P loops (P1 and P2), and four TM spans (TM1–4). Charged residues at position S104 of P1 or T216 of P2 (yellow circles) and second site changes at P131 to G139 in TM2 or G255 to F263 in TM4 (green boxes) are studied. (B) Alignment of K_IR2.1 (amino acids 139–188), K_2PØ P1-TM2 (amino acids 102–149), and K_2PØ P2-TM4 (amino acids 214–273) using ClustalW 1.83. P loops and predicted TM spans are indicated (black lines). The suppressive pore lysine in K_IR2.1 (T141K; yellow circle) and complementing aspartate substitutions in TM2 at C169D, D172, or I176D (green circles) as reported by Chatelain et al. (2005) are indicated. K_2PØ positions are highlighted as in A. (C) KcsA subunit noting sites homologous to those in K_IR2.1 in A and B. (D) Top view of a KcsA channel with one subunit highlighted as in C.