Effects of membrane potentials on the O₁O₂ phenotype. (A) A real-time single-channel recording of N306D upon voltage change from −50 mV (opening: downward deflections) to 50 mV (opening: upward deflections) showing the presence of clearly resolvable O₁ and O₂ states at ±50 mV. The all-points histogram for the recording at 50 mV shows two discernable open states. (B) Opening bursts recorded at 50 mV indicated in A. Note the preferred gating topology of C→O₁→O₂→C is also seen at 50 mV. (C) A representative recording and its all-points histogram showing the existence of the O₁O₂ phenotype at 50 mV for M348R/R352Q. (D) The O₁O₂ phenotype at 50 mV and its all-points histogram for F311R/R303Q/Cysless. A third peak representing a smaller subconductance state is detected in the all-points histogram, which is also observed in all other CFTR mutations, including WT CFTR occasionally. However, the overall gating topology is still dominated by the O₁ and O₂ states. As described in the Materials and methods section, for a clearer data presentation, all single-channel traces recorded at negative membrane potentials were inverted except traces in this figure and Fig. 8 E, where conventional presentations were adopted.