KV1.2 channels inactivate through a mechanism similar to C-type inactivation

Figures

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  Figure 1.. KV1.2 WT channels slow-inactivate incompletely. (A) Currents in response to short voltage-clamp pulses to 50 mV applied from a holding potential of −80 mV and lasting 100 ms. Tail currents are observed upon return to a potential of −30 mV. (B) Voltage dependence of the normalized conductance at the test potential (red circles) or the normalized size of the tail current at the beginning of the −30 mV pulse. The data are the mean and error bars are the SEM from n = 7 oocytes (conductance) or n = 15 (tails). The black curve is the fit of the conductance data to Eq. 2 with parameters Ko = 0.13, zapp = 1.77 eo. (C) Currents in response to long (20-s) pulses from −40 to 80 mV in 20-mV steps. The current slowly decays in amplitude as a result of a slow inactivation process. The decay kinetics can be fitted by a sum of two exponential functions. The fit to Eq. 3 is shown by the continuous blue lines, except the −20 mV trace, which was fitted to a single exponential. (D) Inactivation time constants are plotted as a function of voltage. The filled square symbols are the slow time constant, and the filled triangles are the fast time constant. Amplitudes are shown by empty symbols. (E)…

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  Figure 2.. Effect of extracellular potassium and TEA on slow inactivation in WT KV1.2. (A) Outward currents in response to a 60-mV, 20-s pulse. The blue trace is the normalized average current from eight experiments recorded in 2 mM external K+. The yellow trace is the normalized average current from eight traces recorded in the presence of 100 mM external K+. (B) Quantitation of the effect of potassium on the time constants of inactivation. The color code is the same as in A; squares, slow time constant; triangles, fast time constant. (C) Amplitudes of the fast (inverted triangles) and slow (circles) components are plotted. (D) Effect of external TEA on the kinetics of slow inactivation. The blue trace is the normalized average current at 60 mV from seven traces in the absence of TEA and at 2 mM external potassium. The green trace is the normalized average current from seven sweeps in the presence of 60 mM external TEA also with 2 mM external potassium. (E) Effect of TEA in the slow (squares) and fast (triangles) time constants. (F) Effect of TEA on the amplitudes of the fast (inverted triangles) and slow (circles) time constants. In A and D, the light-colored shade along the current trace…

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  Figure 3.. Mutation W366Y accelerates slow inactivation. (A) Ionic currents in response to 100-ms depolarizing pulses from −80 to 50 mV in 10-mV steps, from a holding potential of −80 mV. Currents are very similar to WT channels. (B) Comparison of the voltage dependence of W366Y and WT KV1.2. The normalized conductance of both channel types is plotted as a function of voltage. Red circles correspond to WT and green circles to W366Y. The continuous curve corresponds to the fit to Eq. 2 with parameters zapp = 1.77 eo, Ko = 0.135 (WT); zapp = 1.59 eo, Ko = 0.149 (W366Y). (C) Mutant channels also inactivate slowly and more completely than WT channels. Blue lines are the fit to Eq. 3. (D) High potassium speeds up inactivation. Green trace is the normalized average current at 60 mV from seven oocytes in the presence of 2 mM external K+. The yellow trace is the normalized current average from eight oocytes in 100 mM external K+. In both cases, the shaded areas represent ± SEM. The black dashed curve is the average WT current time course, also at 60 mV. (E) Voltage dependence of the time course of slow inactivation. Filled symbols are the slow and fast time constants in ND96 extracellular medium…

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  Figure 4.. The W366Y mutant induces a rapidly equilibrating state occurring after the open state. (A) Inside-out patch recording of potassium currents through W366Y channels. Currents were elicited by voltage pulses of 100-ms duration, from −60 to 30 mV from a holding potential of −90 mV. (B) Slow inactivation is also present in cell-free recordings and is incomplete at the end of 5-s pulses. The inside-out data show that inactivation proceeds via two exponentials, comparably to the behavior of currents obtained in TEVC (red curves are fit to a double-exponential function). (C) Nonstationary noise analysis of currents at 20 mV. In the upper panel, 100 current sweeps are shown in red with the mean current plotted in black. The blue trace is the point-by-point variance calculated from the 100 traces. The bottom panel shows the mean–variance relationship (black crosses) with the fit to Eq. 1 shown in red. The parameters of the fit are I = 0.52 pA, n = 6,994. (D) The voltage dependence of activation of WT (red circles) and W366Y (blue squares) channels. The amplitude of the tail current at the end of 100-ms pulses is plotted as a function of the voltage of the pulse and is normalized to the…

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  Figure 5.. The W366F mutation produces channels with fast inactivation. (A) Family of currents showing fast inactivation even with short (100-ms) pulses. Depolarizing pulses stepped from −80 to 50 mV in 10-mV increments, from a holding potential of −80 mV. (B) Comparison of the time course of currents activated by a 100-ms pulse to 50 mV for WT, W366F, and W366Y channels. Currents were normalized to their maximum value. (C) Inactivation time constants for the two mutants and WT channels. The fast and slow time constants for WT (red) and W366Y (green) were obtained from currents elicited by 20-s pulses as in Figs. 1 D and 3 C. The inactivation time constant for W366F (light blue) was obtained from a fit to a single-exponential function of the decay of currents elicited by 100-ms pulses. Data are the mean from nine experiments and error bars are ± SEM. (D) Fast-inactivating currents obtained from an inside-out patch expressing W366F channels. (E) The voltage dependence of these currents is shifted to negative voltages when 60 mM extracellular potassium is used. The continuous black curve is the fit to Eq. 2 with parameters zapp = 1.75 eo, Ko = 0.0091 (n = 6). For comparison, the voltage…

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  Figure 6.. Inactivation characteristics of the W366F mutant. (A) Current elicited by a 50-mV pulse of 100-ms duration obtained in the presence of 2 mM extracellular K+ (continuous line). The dotted line is a representative current at the same voltage in the presence of 100 mM extracellular K+. (B) The inactivation time constant obtained from a single exponential fit to currents in the presence of 2 mM [K+]o (filled symbols) or 100 mM [K+]o (hollow symbols). The black lines show an exponential fit to Eq. 6. The apparent charges in high and low external potassium are similar (−0.17 and −0.19 eo, respectively), and the value of τo is increased from 0.005 to 0.019 s by the high potassium. (C) Effect of external potassium on the voltage dependence of the conductance calculated from the peak current. Filled symbols are the normalized peak conductance in 2 mM [K+]o from n = 9 experiments, and the continuous line is the fit to Eq. 2 with parameters zapp = 1.52 eo, Ko = 0.35. Hollow symbols are the normalized peak conductance in 100 mM [K+]o. The parameters of the fit (dashed curve) are zapp = 1.64 eo, Ko = 0.068 (n = 9). (D) Steady-state inactivation. A prepulse of varying voltage from −120 to −10…

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  Figure 7.. Extracellular and intracellular TEA slows down the inactivation kinetics of the W366F mutant. (A) Representative current traces at 60 mV in the absence (continuous trace) and presence (dashed trace) of 20 mM extracellular TEA. (B) Voltage dependence of the time constant of inactivation. External TEA data are show by hollow symbols, while control data are shown by filled symbols. (C) Effect of intracellular TEA. 20 mM intracellular TEA was applied to inside-out patches. In the presence of TEA, current inactivation is slowed down appreciably at negative voltages. Data shown in B and C are mean ± SEM.

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  Figure 8.. Mutations at V381 in the W366F background produce permanently inactivated channels. (A) Representative traces of gating currents recorded from the expressed channels indicated in each panel. Currents were elicited by voltage-clamp pulses of 60-ms duration ranging from −110 to 30 mV in 10-mV steps from a holding potential of −100 mV. (B) Normalized voltage dependence of charge movement (Q–V) of the three mutants plotted (following its color code from A) and compared with the voltage dependence of the peak-normalized conductance of the background mutation W366F (cyan rhombuses). Q–V data were fitted to Eq. 5. The number of experiments were W366F/V381A, n = 13; W366F/V381T, n = 5; and W366F/V381I, n = 9. (C) The time course of current decay of the three double mutants is plotted as a function of pulse voltage. ON-gating current decay was fitted to a single exponential to determine the time constant. Number of experiments is as in B. Data in B and C are mean ± SEM.

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  (Scheme 1).

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  Figure 9.. Structural basis of modulation of inactivation by mutants at positions W366 and V381. (A) Representation of the area near the selectivity filter of the KV1.2-KV1.1 chimera, showing the residues involved in slow inactivation. The voltage-sensing domain is shown in gray ribbons and the filter domain is rendered in transparent yellow volume and ribbons. Residues W366, Y377, and V381 are shown in red. A TEA molecule is shown in green to indicate the position of the extracellular pore entrance. The blue square indicates the magnified area in B. (B) Left: Residues involved in slow inactivation near the extracellular region of the selectivity filter of the WT KV1.2-KV1.1 chimera. The spheres’ representation in colors corresponds to residues W366, V381, and Y377. Residue D379 is shown in sticks. The dotted yellow line shows the hydrogen bond between W366 and D379. Right: The double mutant W366F/V381A. The main changes in this mutant are an increased space and reduced interaction between W366F and Y377 and elimination of the hydrogen bond between W366 and D379. V381A might enhance the effects of W366F, because it should help the expansion of the space between W366F and Y377. The magenta…