Inactivation as a new regulatory mechanism for neuronal Kv7 channels

Abstract

Voltage-gated K(+) channels of the Kv7 (KCNQ) family have important physiological functions in both excitable and nonexcitable tissue. The family encompasses five genes encoding the channel subunits Kv7.1-5. Kv7.1 is found in epithelial and cardiac tissue. Kv7.2-5 channels are predominantly neuronal channels and are important for controlling excitability. Kv7.1 channels have been considered the only Kv7 channels to undergo inactivation upon depolarization. However, here we demonstrate that inactivation is also an intrinsic property of Kv7.4 and Kv7.5 channels, which inactivate to a larger extent than Kv7.1 channels at all potentials. We demonstrate that at least 30% of these channels are inactivated at physiologically relevant potentials. The onset of inactivation is voltage dependent and occurs on the order of seconds. Both time- and voltage-dependent recovery from inactivation was investigated for Kv7.4 channels. A time constant of 1.47 +/- 0.21 s and a voltage constant of 54.9 +/- 3.4 mV were determined. It was further demonstrated that heteromeric Kv7.3/Kv7.4 channels had inactivation properties different from homomeric Kv7.4 channels. Finally, the Kv7 channel activator BMS-204352 was in contrast to retigabine found to abolish inactivation of Kv7.4. In conclusion, this work demonstrates that inactivation is a key regulatory mechanism of Kv7.4 and Kv7.5 channels.

FIGURE 1

Two-electrode voltage-clamp experiments on Kv7.1, 2, 4, and 5 homomeric channels in Xenopus oocytes (A–D). The channels were activated by voltage steps from −80 mV to potentials ranging from −100 to +60 mV in 20-mV increments (2-s duration), and tail currents were measured at −30 mV. Indications of an inactivation process of Kv7.1 and Kv7.5 can be seen by the small hook on the tail current caused by release from inactivation during the initial phase of the 1-s pulse to −30 mV as well as the marked difference in activation/deactivation kinetics.

FIGURE 2

Magnitude of inactivation. (A–D) The inactivation properties of homomeric Kv7 channel complexes were investigated by voltage-clamp experiments in Xenopus oocytes. After a 20-s-long prepulse at potentials ranging from −120 mV to +60 mV (V_pre), the maximal current amplitudes were measured during a 5-s pulse at +40 mV (V_act) for each channel subtype: A. Kv7.1, B. Kv7.2, C. Kv7.4, and D. Kv7.5 (inset shows the voltage protocol). (E) The degree of inactivation of each Kv7 channel complex is revealed by plotting the current amplitude normalized to the level measured after a V_pre of −120 mV as a function of the preceding V_act potential. Maximal inactivation of ∼80% is observed for Kv7.5 at +60 mV.

FIGURE 3

Time dependence of inactivation. (A) The onset of inactivation of Kv7.4 at 0 mV was studied by a two-step protocol designed to allow the channels to inactivate increasingly. The inset in B shows the voltage protocol employed, which clamped the cell membrane at −120 mV for 20 s to allow for recovery from inactivation (V_pre), followed by a step to 0 mV of 0–28-s duration in 2-s increments. The second step was a depolarizing step to +40 mV for 5 s during which the maximal current amplitude was measured. This experiment was also performed with a 10-s-long V_pre (current traces not shown). (B) Plotting the maximal current amplitude normalized to the amplitude at V_{0 mV, 0 s} as a function of the time spent at 0 mV revealed a single-exponential correlation of the onset of inactivation for both protocols. Fitting the data from the two experiments to I(x) = A_∞ + A × exp(−x/τ) gave significantly different time constants: τ_{10 s prepulse} = 9.55 ± 1.14 s (n = 7) and τ_{20 s prepulse} = 5.06 ± 1.10 s (n = 3); P < 0.05, t-test. The curves are generated from the fitted parameters.

FIGURE 4

Time-dependent recovery from inactivation of Kv7.4. (A) The time course of recovery from inactivation was studied by a two-step protocol in which two activating pulses follow a 20-s-long hyperpolarizing pulse at −120 mV. Each +40-mV pulse (V1_act and V2_act) is of 5-s duration, and the variable parameter is the length of the separating −120 mV hyperpolarizing pulse. The duration of the hyperpolarizing pulse ranges from 250 ms to 3750 ms in 250-ms increments (inset shows the voltage protocol). (B) The maximal current amplitude at V2_act is depicted in percentage of the maximal current amplitude at V1_act subtracting the A₀ (the calculated current amplitude in percentage at time 0) as a function of the release time at −120 mV (i.e., I[V2_act]/I[V1_act] × 100% − A₀), and this revealed a single-exponential relationship between the release time and the degree of recovery from inactivation. Fitting the data to I(x) = A₀ + A × (1 − exp(−x/τ)) gave a time constant τ = 1.47 ± 0.21 s (n = 7). The curve is generated from the fitted parameters.

FIGURE 5

Voltage-dependent recovery from inactivation of Kv7.4. (A) The kinetics of recovery from inactivation was studied by a two-step protocol similar to the one used in the time-dependent recovery experiments, but with the duration of the release pulse fixed at 3 s. The variable in this experiment was the voltage employed to clamp the membrane during the release pulse, which ranged from −120 mV to −30 mV in 10-mV increments (inset shows the protocol). (B) The maximal current amplitude recorded at V2_act in percentage of the corresponding amplitude of V1_act plotted as a function of the holding potential of the release pulse could best be fitted to a single-exponential function: I(x) = A_∞ + A × exp(−x/τ), resulting in a voltage-constant τ = 54.9 ± 3.4 mV (n = 10).

FIGURE 6

Kv7.3+4 heteromers inactivate differently from Kv7.4 homomers. (A) Cells expressing heteromeric Kv7.3+4 channels were investigated for the inactivation behavior of the respective channels with the same voltage protocol as in Fig. 2 (expanded to include a prepulse step to +80 mV). (B) The figure shows the normalized maximal current recorded at a 5-s-long depolarizing pulse to +40 mV preceded by a 20-s-long prepulse (V_pre) ranging from −120 mV to +80 mV in 20-mV increments for both Kv7.4 and Kv7.3 + 4. Current amplitudes are plotted as a function of the holding potential during the 20-s-long prepulse, and the two channel complexes are significantly different in this respect after a prepulse ranging from −40 mV to +60 mV. Asterisks indicate the level of significance (*, P < 0.05; **, P < 0.005, t-test, n = 10 and 27, Kv7.3 + 4 and Kv7.4, respectively).

FIGURE 7

Pharmacological modulation of Kv7.4 inactivation. (A–D) Two-electrode voltage-clamp experiments on three different oocytes under control conditions (A) and after application of 10 μM retigabine (RTG; B), 10 μM BMS-204352 (C), and 100 μM XE-991 (D). The voltage protocol consist of a 20-s-long prepulse (V_pre) ranging from −120 mV to +80 mV in 20-mV increments, followed by a depolarizing step to +40 mV (V_act) and ended by a 1-s step to −30 mV. The scale bars are for control and RTG traces 3 μA and 5 s, for the BMS experiment 6 μA and 5 s, and for XE-991 1 μA and 5 s. (E) Plot showing the normalized current levels before and after application of RTG, BMS, and XE-991, respectively. (F) Graphs illustrate the best linear fit of the measured current expressed as percentage of control data. Best-fit slope values are (in percentage per mV) α_RTG = 0.33 ± 0.21 (n = 7), α_XE-991 = −0.016 ± 0.017 (n = 5), and α_BMS = 5.24 ± 1.51 (n = 3). (G) Histogram illustrating the differences between the slope values determined for RTG, BMS, and XE-991.