Restoration of ΔK_2PØ channel function by second site changes TM2 or TM4. Currents studied as in Fig. 2 A. Bar graphs show current amplitude ± SEM at −100 mV. Sample current traces of channels in which second mutations have no impact or show different restoring effectiveness. Topology insets: blue circles denote S104K in P1 or T216K in P2, and red bars indicate the sites in TM2 or TM4 serially altered to aspartate. (A) S104K ΔK_2PØ channels with no other change or second sites altered to aspartate from 131 to 139 in TM2. Statistical differences versus S104K are indicated (n = 9). A helical wheel plot indicates four TM2 sites that restore function (133, 134, 137, and 138). (B) T216K ΔK_2PØ channels with no other change or second sites altered to aspartate from 255 to 263 in TM4. Statistical differences versus T216K are indicated (n = 9). A helical wheel plot indicates four TM4 sites that restore function (257, 258, 261, and 262). (C) S104K ΔK_2PØ channels with no other change or second sites altered to aspartate from 255 to 263 in TM4. Statistical differences versus S104K are indicated (n = 10). A helical wheel plot indicates two TM4 sites that restore function (258 and 262). (D) T216K ΔK_2PØ channels with no other change or second sites altered to aspartate from 131 to 139 in TM2. Statistical differences versus T216K are indicated (n = 5–10). A helical wheel plot shows three TM2 sites that restore function (134, 138, and 139). *, P < 0.05; **, P < 0.01; ***, P < 0.001.