Restoration of ΔK_2PØ–ΔK_2PØ channel currents by second site changes in the same and adjacent domains. Currents suppressed by S104K in domain I of ΔK_2PØ–ΔK_2PØ are restored by second site substitution of aspartate in domains I, II, and IV but not III. Similarly, currents suppressed by T216K in domain II of ΔK_2PØ–ΔK_2PØ are restored by second site substitution in domains I, II, and III but not IV. Currents were studied as in Fig. 2 A. (A) Predicted topology of ΔK_2PØ–ΔK_2PØ with S104K in domain I (blue circles); domains I–IV are indicated, as are TM spans with second site changes (red bars). (B) Currents from ΔK_2PØ–ΔK_2PØ channel variants. To reveal phenotype, oocytes with WT–WT, S104K (I)–WT, and S104K (I)–S104K (III) were incubated for extended periods. (C) Current amplitudes ± SEM at −100 mV from oocytes expressing S104K (I) ΔK_2PØ–ΔK_2PØ channels with no other change or second sites altered to aspartate as indicated. Statistical differences versus S104K (I) ΔK_2PØ–ΔK_2PØ are indicated (n = 6). (D) Top view of a KcsA channel noting positions homologous to S104K in domain I (blue) and TM segments studied in each of four domains (red). (E) Predicted topology of ΔK_2PØ–ΔK_2PØ with T216K in domain II (blue circles); domains I–IV are indicated, as are TM spans with second site changes (red bars). (F) Representative currents from the indicated ΔK_2PØ–ΔK_2PØ channel variants. (G) Current amplitudes ± SEM at −100 mV from oocytes expressing T216K (II) ΔK_2PØ–ΔK_2PØ channels with no other change or second sites altered to aspartate as indicated. Statistical differences versus T216K (II) ΔK_2PØ–ΔK_2PØ are indicated (n = 14–16). (H) Top view of a KcsA channel noting positions homologous to T216K in domain II (blue) and TM segments studied in each of four domains (red). *, P < 0.05; **, P < 0.01; ***, P < 0.001.