Figure 3. Inactivation of the Shaker-IR T449K channel cannot trap internally applied bTBuA.

The mutant Shaker-IR T449K was heterologously expressed in Xenopus oocytes as explained under Methods. (a) Inside-out macropatch outward currents evoked by a step to +50 mV from a holding voltage of −100 mV. The overlaid traces depict the current profile before (black), during (blue) and after (green and gray) exposure of the intracellular side of the channel to 100 μM bTBuA. The exposure to the QA ion began before the step depolarization and was terminated after macroscopic inactivation reached steady-state (Fig. 2). Note that no exposure was allowed during the repolarizing step that closes the channels. (b) Magnitude of peak currents in two consecutive iterations of the experiment described above. Pulses P1 (Control, C), P2 (Block, B) and P3–P8 evoke the currents as explained in Fig. 2. (c) Scaled and normalized currents from panel (a). (d) Time constants of decay from the currents evoked by the test pulses as explained above for panel (a). Generally, one-two exponentials were sufficient to describe this decay. When the sum of two exponentials yielded the best fit, the reported time constant is the weighted average of the best-fit time constants. (e) Box plots of the time constants of current decay during blockade by bTBuA (blue box), after washout (green box) and from the currents evoked by P8 (N = 5 patches; 1–3 iterations each). (f) Box plots of the relative peak current amplitudes during blockade by bTBuA (blue box), after washout (green box) and from the currents evoked by P8 (N = 5 patches; 1–3 iterations each). On average, the peaks of currents evoked by P3 and P8 are not significantly different (Kruskal-Wallis, p = 0.37).