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. 2018 Mar 26;115(15):E3559–E3568. doi: 10.1073/pnas.1717082115

Fig. 3.

Fig. 3.

V404M dramatically slows inactivation after opening but accelerates inactivation from closed states. (A, Upper Left) To characterize the kinetics of inactivation from partially or fully activated closed states, the membrane was stepped from −100 mV to prepulse voltages from −80 to −20 mV for durations from 0 to 1,056 ms before a test pulse to +20 mV. [A, Upper Center (wild-type Kv4.2) and Upper Right (V404M)] Current amplitude at +20 mV was normalized to the current amplitude obtained in the absence of a prepulse (i.e., Δt = 0) and plotted vs. Δt. Representative data obtained after a prepulse to −60 mV are shown. The data were fitted with a single exponential function to estimate the inactivation time constant, τinact. Fits are shown superimposed on the data. (A, Lower Left) To characterize the kinetics of inactivation after opening, the membrane was depolarized from −100 mV to voltages from −50 to +90 mV for 1.5 s (wild-type Kv4.2) or 21.5 s (V404M). [A, Lower Center (wild-type Kv4.2) and Lower Right (V404M)] Representative current traces recorded at +20 mV are shown. Macroscopic current decay was fitted with a single exponential function to estimate the inactivation time constant, τinact. Fits are shown superimposed on the data (dashed gray curves). (B and C) Values of τinact for (B) wild-type Kv4.2 and (C) V404M obtained using the closed-state (■) and open-state (blue ●) protocols have been plotted on a log scale vs. prepulse or step voltage, respectively. Values are provided as mean ± SEM (wild-type, n = 5–14; V404M, n = 4–10). Both protocols were applied at prepulse or step voltages from −50 to −20 mV (dashed boxes). (B) In wild-type channels, values of τinact obtained at −20 mV from the closed-state (70 ± 4 ms) and open-state (79 ± 1 ms) protocols differed significantly (n = 10). *P < 0.05. (C) In V404M channels, values of τinact obtained by the closed- and open-state protocols differed significantly from −50 to −20 mV (P < 0.00001, n = 5–10). (D) Values of τinact obtained using the closed-state protocol for wild-type Kv4.2 (■; n = 5–12) and V404M (red ●; n = 7–10) have been plotted vs. prepulse voltage. Values of τinact, provided as mean ± SEM, differed significantly at all voltages. †P < 0.0001; ‡P < 0.00001. WT, wild type.