Figure 8.
Figure 8. O1 and O2 states exhibit differential sensitivity to channel-impermeant blocker MOPS−. Current traces filtered at 100 Hz are shown as a gray background, whereas the same traces filtered at 10 Hz are colored in blue and super-positioned. For the traces in A–D, 30 mM MOPS− was applied in the presence of 2 mM ATP in the early part of the recordings and then removed from the ATP-containing solution at the time point indicated by the arrowheads. The all-points histograms for the traces in the absence of MOPS− are shown as blue curves, and the histograms for the traces in the presence of MOPS− are shown as black curves. Dashed lines represent the baselines. (A) A representative trace of WT CFTR in the presence or absence of MOPS−. As reported previously (Ishihara and Welsh, 1997), two distinct open-channel conductance levels are clearly discerned once the data are filtered at 10 Hz. (B) Similar to WT CFTR that has only one resolvable open-channel conductance level in the presence of ATP, F311C/Cysless also shows two distinct open-channel conductance levels once blocked by MOPS−. (C) Amplification of the difference in single-channel amplitude between O1 and O2 states by MOPS− in M348R/R352Q, which itself exhibits the O1O2 phenotype. The normalized difference (Δi/iO1) is 51 ± 2% without MOPS− (n = 9) but 101 ± 2% with MOPS− (n = 4). (D) Effects of net charge in the internal vestibule on the differential sensitivity of O1 and O2 states to MOPS− block. Contrary to the results in A–C, N306D with one additional negative charge in the internal vestibule displays the O1O2 phenotype in the presence of ATP, but its O1 and O2 states show similar sensitivity to MOPS− block. The O2 states of N306D in the presence of MOPS− could not be resolved by the all-points histogram because of its small single-channel amplitude and the brief open time of the O2 state. Similar to N306D, R303C/Cysless also has one net charge deducted from the internal vestibule of CFTR’s pore. For this mutant, however, only one open-channel level is seen in the absence or presence of MOPS−. (E) Compared with the result in B, the representative trace of F311C/Cysless recorded at 50 mV in the presence of 2 mM ATP shows negligible MOPS− block.

O1 and O2 states exhibit differential sensitivity to channel-impermeant blocker MOPS−. Current traces filtered at 100 Hz are shown as a gray background, whereas the same traces filtered at 10 Hz are colored in blue and super-positioned. For the traces in A–D, 30 mM MOPS− was applied in the presence of 2 mM ATP in the early part of the recordings and then removed from the ATP-containing solution at the time point indicated by the arrowheads. The all-points histograms for the traces in the absence of MOPS− are shown as blue curves, and the histograms for the traces in the presence of MOPS− are shown as black curves. Dashed lines represent the baselines. (A) A representative trace of WT CFTR in the presence or absence of MOPS−. As reported previously (Ishihara and Welsh, 1997), two distinct open-channel conductance levels are clearly discerned once the data are filtered at 10 Hz. (B) Similar to WT CFTR that has only one resolvable open-channel conductance level in the presence of ATP, F311C/Cysless also shows two distinct open-channel conductance levels once blocked by MOPS−. (C) Amplification of the difference in single-channel amplitude between O1 and O2 states by MOPS− in M348R/R352Q, which itself exhibits the O1O2 phenotype. The normalized difference (Δi/iO1) is 51 ± 2% without MOPS− (n = 9) but 101 ± 2% with MOPS− (n = 4). (D) Effects of net charge in the internal vestibule on the differential sensitivity of O1 and O2 states to MOPS− block. Contrary to the results in A–C, N306D with one additional negative charge in the internal vestibule displays the O1O2 phenotype in the presence of ATP, but its O1 and O2 states show similar sensitivity to MOPS− block. The O2 states of N306D in the presence of MOPS− could not be resolved by the all-points histogram because of its small single-channel amplitude and the brief open time of the O2 state. Similar to N306D, R303C/Cysless also has one net charge deducted from the internal vestibule of CFTR’s pore. For this mutant, however, only one open-channel level is seen in the absence or presence of MOPS−. (E) Compared with the result in B, the representative trace of F311C/Cysless recorded at 50 mV in the presence of 2 mM ATP shows negligible MOPS− block.

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