FIGURE 2.

Irreversibility of CuPhe inhibition in double cysteine mutants. A, example whole cell currents recorded at +30 mV for T338C/S1118C from cells that had been pretreated with CuPhe either together with FSK (left panel) or without FSK (right panel) for 5 min. Following extensive washing and attainment of the whole cell patch configuration, channel activation by application of cAMP stimulatory mixture revealed currents that were increased by DTT application (5 mm) only if CuPhe was applied together with FSK. In each case, the identity of CFTR currents was confirmed using the specific inhibitor GlyH-101 (50 μm). B, mean effect of DTT application on whole cell current amplitude for the three double cysteine mutants named, following different pretreatment conditions. Asterisks indicate a significant difference from no pretreatment conditions (p < 0.01), and daggers indicate a significant difference from pretreatment with CuPhe in the absence of FSK (p < 0.01). The means of data from three to six cells are shown. C, stability of CuPhe inhibition of R334C/G1127C and reversibility by excess DTT. Example whole cell current from a cell treated sequentially with cAMP stimulatory mixture, CuPhe, DTT, and GlyH-101.