Measurements of the second-order reaction rate reveal that TM12 assumes an α-helical structure. (A and B) Measurements of the second-order reaction rate constant (κ_MTSES⁻) of MTSES⁻ modification in the presence of 2 mM ATP for cysless/Q1144C (A) and cysless/S1141C (B). Single-exponential fitting (red line) of the current relaxation in response to MTSES⁻ treatment yields the modification time constant (τ), the reciprocal of which divided by the concentration of MTSES⁻ gives κ_MTSES⁻. (Bottom traces I and II) Expanded view of portions of top traces (as marked) to show that the single-channel amplitude is diminished after MTSES⁻ exposure. (C) Summary of the modification rate constant at each position of the cytoplasmic half of TM12. Note that reaction rate at positions 1141, 1145, and 1148 approximates that of MTSES⁻ with free thiols (green line). Crossed circles mark cysteine-substituted channels whose function was not significantly altered by the treatment of MTSES⁻. The number near each bar represents the number of patches. (D) Helical structure of the cytoplasmic half of TM12 inferred from C. Blue, positions where introduced cysteines were readily accessible to MTSES⁻; light blue, positions where cysteines were less accessible; green, positions where no accessibility was observed.