Figure 6. Inactivated Shaker–IR T449K allows the access of bTBuA to the pore and the blocker gets trapped in the pore upon hyperpolarization.

T449K channels were transiently expressed in ts-A201 cells (see Methods). (a) Voltage- and concentration-clamp protocol to probe for bTBuA ion trapping by inactivation. (b) Macroscopic outward currents were recorded in inside-out patches. The patches were repeatedly depolarized from a holding potential of −100 to +50 mV for 5 s to ensure complete inactivation. The nomenclature of the pulses in the sequence is in panel a. The interpulse interval was 5 s and the patches were held at the holding potential between the pulses. The intracellular side of the patch was exposed to 100 μM bTBuA for 1.5 s (1 s at +50 mV and 500 ms at −100 mV). The timing and the duration of the bTBuA pulse are indicated by the solid blue bar. The overlaid traces depict the currents recorded in control solution (P1, C=control, black), during the first pulse following the exposure of the intracellular side of inactivated channels to 100 μM bTBuA (P2, B=block, blue) and during subsequent pulses in control solution (P3, green, and P4–P8, grey) (only 100-ms-long segment of the currents is shown for clarity). (c) Scatter plots of the normalized peak current amplitudes measured during pulses C, B, and P3–P8 (see above). Horizontal bars indicate the mean of N experiments. (d) Currents in panel b were normalized to their respective peak and shown as a function of time. Color code is the same as in panel a. (e) Scatter plot of the individual time constants obtained from currents evoked by pulses C, B, and P3–P8 (see above) and the mean of the time constants (horizontal bars). Inactivation time constant of the current at +50 mV was determined by fitting a single exponential function to the decaying part of the currents.