Figure 4.

Complete blockade of P_C by AgNO₃. (A and B) Full blockade of P_C after the addition of AgNO₃ (cis, 33 nM free Ag⁺ ion). Soon after the appearance of reversible Ag⁺ ion binding events as shown in Fig. 3A, P_C underwent a stepwise decrease in conductance, which eventually led to complete blockade (64% of experiments exhibited full blockade). The applied potential was switched to +50 mV as indicated by black arrows on the current traces. In A, a sudden large drop in conductance was first observed, followed by a stepwise decrease in conductance. In B, a stepwise decrease in conductance from the open pore level to the fully blocked level was seen. The noise in the intermediate levels may be caused by polymer movement (35,36). (C and D) Reopening of blocked P_C. After the full blockade induced by AgNO₃, cysteamine was added to the cis compartment. More than one equivalent of cysteamine (10 μM) relative to the total amount of AgNO₃ (cis, 8 μM) was necessary for unblocking. Cysteamine forms oligomers with Ag⁺ ion in solution (37). The oligomers occasionally react with the protein cysteine residue, leading to additional incomplete blockades. Panels C and D show one-step and stepwise unblocking, respectively.