Figure 3.

Gating current measurements of Shaker-IR-W434F and P475A. (A) Gating current recordings of Shaker-IR-W434F control obtained with activation (left) or a deactivation (right) protocol. On the left, a scaled up view of the activating gating currents (I_Qac) at +20 mV (blue) and +60 mV (red) are shown as inset. With increasing depolarization strengths the I_Qac kinetics accelerate causing the I_Qac tracings to cross each other. Inset on the right shows a scaled up view of the deactivating gating currents (I_Qdeac) at −90 mV (blue) and −120 mV (red). Similar to I_Qac, I_Qdeac decay accelerated with stronger repolarizations. (B) Representative I_Qac (left) and I_Qdeac (right) recordings of P475A. Insets show a scaled up view of both I_Qac and I_Qdeac obtained with the activation protocol. Similar to control (panel A), I_Qac accelerated with stronger depolarizations, which is highlighted by coloring the recordings at 0 mV (green), +50 mV (blue), and +130 mV (red). Scale up view of the I_Qdeac recordings at −90 mV shows that I_Qdeac did not slow down up to prepulse depolarizations of +50 mV in strength (note the overlap of the green and blue recording, which were obtained upon a 0 and +50 mV prepulse, respectively). However, upon a +130 mV prepulse (red trace) there was a reduction in I_Qdeac amplitude suggesting that the I_Qdeac kinetics were slowed down (larger scale of this scaled up view is shown in the Supporting Material). Right panel displays I_Qdeac recordings that were elicited with a deactivation pulse protocol by stepping after a 150-ms +90 mV prepulse to potentials between 0 and −120 mV. The blue and the red trace were obtained at −80 mV and −120 mV, respectively. (C) QV curves (symbols and line, which represent average fit with a single or sum of two Boltzmann distributions) of Shaker-IR-W434F (black) and P475A (blue) obtained by integrating and normalizing the I_Qac recordings. For comparison, the GV curves are represented in dotted lines. Note, the QV curve of P475A displayed two gating charge components whereby the first component matched the QV curve of Shaker-IR-W434F, whereas the second component was shifted by ∼ +95 mV and corresponded to the shift in the GV curve. (D) Voltage-dependency of the I_Qac (circles) and I_Qdeac (triangles) time constants ± SE for Shaker-IR-W434F (gray symbols, n = 8) and P475A (blue symbols, n = 6). (E) Envelope pulse protocol to determine in P475A the effect of prolonging the prepulse depolarization on the speed of I_Qdeac decay (τ_I_Qdeac), which was elicited by repolarizing to −100 mV. With longer depolarization times at +100 mV there was a gradual decrease in I_Qdeac amplitude, most likely because of a slowing down in τ_I_Qdeac. Approximating the decay in I_Qdeac amplitude with a single exponential function (blue dotted line) yielded a time constant of 58.8 ms in the represented recording.