E116 forms a salt bridge with R104 in both the closed and open states. (A) Representative single-channel current traces of R104E- and R104E/E116R-CFTR recorded with the same experimental conditions as Fig. 2 (left), their all-points amplitude histograms (middle), and mean burst durations (right). **, P < 0.01 indicates a significant difference between E116R- and R104E/E116R-CFTR. (B) Two cysteines engineered at positions 104 and 116 (R104C/E116C) form a spontaneous disulfide bond when CFTR is in the open state. Representative single-channel trace of R104C/E116C-CFTR recorded with the same conditions as A. Control, 150 mM Cl⁻ extracellular solution alone (left, top trace). DTT, dithiothreitol reducing agent. +DTT in pipette: R104C/E116C-CFTR recorded with 1 mM DTT in the extracellular pipette solution (left, middle trace). In the bottom trace, oocytes expressing R104C/E116C-CFTR were incubated in solution containing 5 mM MTSET⁺ over 10 min before single-channel current recording (+MTSET; left, bottom trace). Their all-points amplitude histograms are shown in the middle panel. Mean fraction of open burst duration is plotted at right for R104C/E116C-CFTR under three different experimental conditions, for each of the open conductance states: s1, dark red; s2, orange; and f, light green. (C) Cross-linking R104C to E116C using MTS-2-MTS locks CFTR channels into the closed state. Representative trace (left) and summary data (right) for macroscopic currents measured from R104C/E116C-CFTR with addition of 1 mM MTS-2-MTS in the absence of ISO at V_M = −60 mV. ND96, control bath solution. Current levels in the summary data chart are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1. #, P < 0.001 compared with ISO1 in n = 4 experiments. Mean ± SEM is shown.