Figure 2. Voltage- and concentration-clamp protocol to probe for QA ion trapping by inactivation.

Inside-out macropatches were subjected to eight 5-s depolarizing pulses (P1–P8, −100 to +50 mV) delivered at intervals of 5 s (10-s duty cycle). P1 (C) evokes the control current. P2 (B) evokes the current while the QA ion is applied to the internal side of the inside-out patch using a High Speed Solution Exchange System (HS-SES, Methods). The timing of the application in relation to P2 is shown in the boxed inset. Note that the exposure occurs while the channels undergo activation, opening and inactivation; and that this exposure is terminated 500 ms before the repolarization that closes the channels (once inactivation is complete and at steady-state). This duration is ~20-times longer than the exchange time of the solution switching system (Methods) and, therefore, it is unlikely that QA ions are still present in the intracellular bath solution at the time the channels close by repolarization. Following washout, P3–P8 are then used to test the recovery of the currents. P2, P3 and P4–P8 probe the block, trap and escape stages of the experiment, respectively. To confirm the reproducibility of the results, the P1–P8 sequence and QA ion application were generally repeated as many times as possible until the patch deteriorated.