Cysteine scanning of TM12 of CFTR using intracellularly applied MTSES⁻ reagents defines a cytoplasmic water-accessible region. (A) Current recorded at −50 mV in inside-out membrane patches containing thousands of cysless/S1141C-CFTR channels. Application of a saturating concentration of ATP (black lines and labels) elicited chloride currents (downward deflections). Upon intracellular MTSES⁻ treatment (red lines and labels), the ATP-induced current decreased. Dotted line denotes the baseline. (B) Representative current recording of cysless/I1139C-CFTR channels using a similar experimental protocol as in A. Channel activity is not substantially altered by MTSES⁻ treatment. (C) Summary of the percentage change in macroscopic current amplitude after the intracellular application of MTSES⁻ for each cysteine-substituted channel. The dotted line marks the position beyond which no reactivity toward the cytoplasmically applied MTSES⁻ was observed. The number near each bar marks the number of patches. Data points in this and all other figures are mean ± SEM.