State-dependent modifications of a larger MTS reagent, Texas red MTSEA⁺, suggest that the size of the inner pore entrance in the open state is smaller than that in the closed state. (A) Chemical modification of cysless/S1141C-CFTR channels by Texas red MTSEA⁺ (orange arrows) without ATP. (B) Normalized current decay produced by Texas red MTSEA⁺ with (continuous traces) or without (closed squares) ATP. Single-exponential fits (orange lines) yield the modification time constant (τ). Note that modification by Texas red MTSEA⁺ without ATP is faster than that with ATP. (C) Second-order rate constants (κ_MTSES⁻) of Texas red MTSEA⁺ modification for cysless/S1141C-, cysless/N1148C-, cysless/I344C-, and cysless/M348C-CFTR channels. Positions 1141, 1148, 344, and 348 are located at the cytoplasmic and deep positions of TM12 and TM6, respectively. They are exposed to the watery pore as inferred from the observation that cysteines at these positions are rapidly modified by MTSES⁻. Note that modification by Texas red MTSEA⁺ without ATP is faster than that with ATP for all four mutant channels. (D) Channel activity is minimally altered after the prolonged (∼1-min) treatment of 100 µM Texas red MTSEA⁺, but immediately diminished upon exposure to 50 µM MTSES⁻. Given that the E1371Q mutant channel has an open probability of near unity in the presence of ATP, this result suggests that 1141C is readily accessible to MTSES⁻ but not to Texas red MTSEA⁺ in an open channel. Similar results were observed for cysteines substituted into positions 1148, 344, and 348 under the E1371Q background. Each experiment was repeated in three to six patches.