Cysless/R352C-CFTR reveals two different open states with distinct conductance level. (A) Five representative traces and amplitude histograms from a patch that contained only one Cysless/R352C-CFTR channel. The channel was fully phosphorylated (this also applies to Figs. 3–7), and the traces were recorded in the presence of 2.75 mM ATP. (B) Four representative traces and amplitude histograms for Cysless/R352C-CFTR recorded in a condition similar to that in A, except that both pipette and perfusion solution contain 375 mM Cl⁻ (see Materials and methods for details). High Cl⁻ solution significantly augmented the single-channel amplitude (0.17 ± 0.01 pA and n = 8 in 150 mM solutions vs. 0.38 ± 0.01 pA and n = 11 in 375 mM; holding potential = −60 mV for the larger conductance state). The events in the red boxes show a preferred order of gating transitions (i.e., C→O1→O2→C) and are expanded in Fig. 4 for clearer view. Events after the C→O1→C transition are shown in the green boxes. One event showing the C→O2→C transition is highlighted in the blue box.