Mutagenesis scan of S6 segment identifies residues important for normal Slo2.1 channel gating. (A) 33 consecutive residues (Thr252 to Arg284) located in the S6 segment were individually mutated to Ala (native Ala residues were mutated to Cys), and the effect of 1 and 6 mM NFA on ISlo2.1 was quantified at a test potential of 0 mV. Oocytes were injected with 0.2–2 ng cRNA and recorded 1–3 d later (n = 4–6). Five mutations (K256A, I263A, P271A, Q273A, and E275A) caused complete loss of channel function (indicated by the asterisks). (B) Plot of peak currents measured at 0 mV in the absence (black boxes) and presence of 1 mM NFA (red circles) and 6 mM NFA (blue triangles) for the five loss-of-function mutant channels. Mutant channel currents measured in presence of 6 mM NFA were corrected by subtraction of the mean value for endogenous currents activated by 6 NFA in uninjected oocytes. Oocytes were injected with 10 ng of cRNA and incubated for 3–4 d (n = 6–10). The right panel shows the same data plotted on an expanded y axis. Error bars indicate mean ± SEM.
