Intragenic rescue of nonfunctional E275A Slo2.1 was achieved by introducing a charged residue one helical turn away in the S6 segment. Mean I-V_t relationships recorded before (control) and after treatment of oocytes with NFA for E275A/A278E channels (A; n = 5; 5 ng cRNA injected, currents recorded after 4 d), E275A/A278R channels (B; n = 8; 1 ng cRNA injected, currents recorded after 2 d), and E275A/Y279E channels (C; n = 6; 10 ng cRNA injected, currents recorded after 4 d). (D–F) E275A/Q276E channels (D), E275A/L277E channels (E), and E275A/L280E channels (F). For D–F: n = 6; 10 ng cRNA injected, currents recorded after 5 d. Error bars indicate mean ± SEM. (G) Helical wheel plot of S6 segment of Slo2.1 from Pro271 to Arg284. The arrow points to Glu275, R indicates the two residues that when mutated to Glu rescued E275A channel function, and NR indicates the three residues that when mutated to Glu did not rescue E275A channel function. A wheel plot was made using the Wheel.pl program written by D. Armstrong and R. Zidovetzki (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). Residues are shape- and color-coded to indicate physical properties as follows: circles, uncharged hydrophilic (proportional to the intensity of red color); diamonds, hydrophobic (proportional to the intensity of green color); triangles, acidic; pentagon, basic. (H and I) MthK-based model of the Slo2.1 pore domain (S5-PH-S6) viewed from the intracellular side. Key residues are labeled for one subunit in each panel. Mutation of Ala278 or Tyr279 (H) to Glu rescued function of E275A channels. Mutation of Gln276, Leu277, or Leu280 (I) did not rescue E275A channels.