Molecular rearrangements in S6 during slow inactivation in Shaker-IR potassium channels

Figures

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  Figure 1.. Structure of the pore region and the four-state gating model of Shaker-IR. (A) The structure of the pore domain of Shaker-IR channel (amino acid residues 390-489) in open conformation (PDB accession no. 7SIP) visualized by PyMol. The S5, S6, pore helix, and pore loop segments of two diagonally opposed subunits are shown as ribbon representations with front and rear subunits removed for clarity. Colored amino acids are T449 (red), A471 (purple), L472 (cyan), and 473 (blue). Potassium ions in the selectivity filter are illustrated by black dots. (B) Helical wheel projection of the pore domain (residues 469–479). Amino acid residues indicated by * at the right side of the helical wheel are facing the water-filled cavity in the open state. Color-coding indicates amino acid characteristics, yellow: hydrophobic; gray: polar. (C) Composite gating states and a simplified four-state gating model in which four major gating states can be distinguished depending on the status of the activation (lower) and inactivation (upper) gates. Among the closed (C), open (O), open-inactivated (OI), and closed-inactivated (CI) states, only the open state is conducting. Arrows indicate the theoretically…

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  Figure S1.. Characterization of the kinetics of perfusion exchange. (A) The inside-out patches were held at −120 mV and positioned in the center barrel of the fast perfusion system (see Materials and methods for details). Patches were exposed to 150 mM K+ (hatched) at the holding potential and then depolarized to +50 mV for 200 ms with a concomitant switch of the barrel to one containing 50 mM K+ (dotted). The duration of the exposure to 50 mM K+ was 200 ms. The kinetics of the solution change is depicted in the current trace as a rapid decrease in current. The mechanical and electrical delay is d and the fit of a single exponential to the falling phase of the current (red) gives the time constant of the solution exchange, τe. (B) Violin plots of d and τe show the average values of 24.9 ± 0.7 ms and 10.9 ± 1.0 ms, respectively (n = 30, biological replicates).

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  Figure 2.. Characterization of K+ currents of the T449A/A471C Shaker-IR mutant. (A) Ionic currents of T449A/A471C channel expressed in tsA201 cells. The inside-out patch was held at −100 mV and depolarized to test potentials ranging from −80 to +70 mV in steps of 10 mV every 60 s. The duration of the depolarizing pulses was 1 s. Inset shows K+ currents evoked by the depolarizing pulses to the indicated membrane potentials. (B) Normalized G–V data (see Materials and methods) were obtained from the peak currents (Ipeak) at the test potential of Em and the K+ reversal potential (EK) using G(V) = Ipeak/(Em – EK). The G(V) values were normalized for the maximum conductance (G/G0) for six independent measurements, averaged, and then plotted as a function of test potential. The superimposed solid line is the best-fit Boltzmann function to the averaged data points. The mean values ± SEM of the midpoint (V1/2) and slope factor (s) are shown. (C) Activation kinetics were studied using 5-ms-long depolarizing pulse from a holding potential of −120 to +50 mV. Current traces were fitted using the Hodgkin-Huxley n4-model, and the activation time constant (τact) was used to describe the activation…

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  Figure S2.. Biophysical characterization of K+ currents evoked in T449A/L472C Shaker-IR channel. (A) The inside-out patch was held at −100 mV and depolarized to test potentials ranging from −80 to +70 mV for 1 s in steps of 10 mV every 60 s. Inset shows K+ currents evoked by the depolarizing pulses at the indicated test potentials. (B) Normalized G–V data were obtained from the peak currents (see Materials and methods for details, averaged, and plotted as a function of test potential). The superimposed solid line shows the best-fit Boltzmann function. The mean values ± SEM of six independent measurements of the midpoint (V1/2) and slope factor (s) are shown. (C) Activation kinetics were studied using 5-ms-long depolarizing pulse from a holding potential of −120 to +50 mV. Current traces were fitted using Hodgkin–Huxley n4 model, and the activation time constant (τact) was determined. Inactivation time constant (τi) of the current at +50 mV was determined by fitting a single exponential function to the decaying part of the currents shown in A. Violin plots indicate Q1, median, and Q3 of τact and τi (n = 23), symbols indicate individual data points. (D) Voltage dependence of steady-state…

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  Figure S3.. Biophysical characterization of heteromeric T449A/P473C//T449 Shaker-IR channel. (A) Macroscopic K+ currents evoked by 1-s-long test potentials ranging from −80 to +70 mV in 10 mV increments every 60 s. Inset shows K+ currents recorded at the indicated test potentials. (B) Averaged normalized G–V data points (see Materials and methods for further description) were plotted as a function of test potential. The superimposed solid line shows the best-fit Boltzmann function. The mean values ± SEM of the midpoint (V1/2) and slope factor (s) are shown (n = 6). (C) Activation kinetics were studied using 10-ms-long depolarizing pulse from a holding potential of −120 to +50 mV. Current traces were fitted using Hodgkin–Huxley n4 model, and the activation time constant (τact) was determined. Inactivation time constants of the current at +50 mV were determined by fitting a double exponential function to the decaying part of the currents shown in A to give time constants of the fast and slow components of slow inactivation, respectively. Violin plots indicate Q1, median, and Q3 of τact, τi,f and τi,s (n = 25), and symbols indicate individual data points. (D) Voltage dependence of steady-state…

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  Figure 3.. Application of MTS reagents to the closed channels. The pictograms on the left indicate the status of the activation (lower) and inactivation (upper) gates. (A) The K+ current was measured from a voltage-clamped inside-out patch containing T449A/A471C channels. Channels were held closed at −120 mV, and their activity was monitored using brief depolarizing pulses to +50 mV every 15 s to measure the K+ current. The patch was exposed for L = 500 ms to 2 mM MTSEA (red hatched bar, MTSEA) and then washed out before the test pulse (lag time = 600 ms). (B–D) Normalized current as a function of cumulative modification time of the MTS reagents: 2 mM MTSEA (red symbols) or 0.2 mM MTSET (green symbols) in the ICS for T449A/A471C (B), T449A/P473C//T449 (C), and T449A/L472C (D). Data were obtained using the protocol explained in A. Filled circles, T449A/A471C; triangle up, T449A/L472C; triangle down, T449A/P473C//T449. Mean ± SEM for at least n = 4 (biological replicate) experiments are shown throughout the figure.

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  Figure 4.. Application of MTS reagents to the open channels. (A, C, and E) The pictograms on the left indicate the status of the activation (lower) and inactivation (upper) gates. Depolarizing pulse durations were 5 ms for T449A/A471C (A, 471C) and for T449A/L472C (C, 472C) or 10-ms-long for the T449A/P473C//T449 heteromeric construct (E, 473C). Depolarizing pulses were delivered from the holding potential of −120 to +50 mV every 15 s in the presence of 2 mM MTSEA (red symbols) or 0.2 mM MTSET (green symbols) in the ICS. The perfusion with MTS reagents started 200 ms prior to the application of and during the depolarizing pulse. The patches were perfused with MTS-free ICS (see Materials and methods) for the duration of the interpulse holding potential. (B, D, and F) Peak currents for each pulse in the presence of MTS (I(t)) were normalized to the last peak current prior to MTS application (I0) and plotted as a function of cumulative modification time (t). Mean ± SEM for at least n = 3 (biological replicates) experiments are shown throughout the figure.

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  Figure S4.. Application of 2 mM MTSEA to T449A/A471C Shaker-IR channel in the open state. K+ currents were evoked by 5-ms-long depolarizing pulses from the holding potential of −120 to +50 mV repeated every 10 s in the presence of 2 mM MTSEA in the ICS. For the composition of the solutions see Materials and methods. (A) Peak currents for each pulse in the presence of MTSEA (2 mM MTSEA, red symbols; I(t)) were normalized to the last peak current prior to MTSEA application (I0), averaged, and plotted as a function of cumulative modification time (t). Control currents were recorded in the absence of MTSEA in the ICS, peak currents (I(t)) were normalized to the peak current (I0) at the beginning of the pulse sequence, averaged, and plotted as a function of cumulative modification time (t; black symbols). (B) Normalized peak currents determined at the 60th depolarizing pulse (identical to 300 ms cumulative modification time in 2 mM MTSEA). Symbols indicate individual data points obtained as control (black symbols) and in the presence of 2 mM MTSEA (red symbols). Data are given as mean ± SEM (n ≥ 6), n values are biological replicates in all panels.

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  Figure 5.. Application of MTS reagents to the inactivated T449A/A471C channels. The pictograms on the left indicate the status of the activation (lower) and inactivation (upper) gates. Patches were repeatedly depolarized from a holding potential of −120 mV using the pulse protocols shown above the corresponding raw current traces. The timing and the duration of the MTS pulses in ICS are indicated by the hatched bars. (A and B) 2 mM MTSEA modifies T449A/A471C channels. (A) 1.0-s-long depolarizing pulses were applied every 60 s and perfusion with 2 mM MTSEA (duration (L) = 500 ms) was initiated 300 ms after the start of the depolarization. (B) Peak currents for each pulse (I(t)) were normalized to the peak current of the last pulse prior to MTS application (I0), averaged, and plotted as a function of cumulative modification time (t). The superimposed line is the best fit for a single exponential function. The reciprocal of the time constant is divided by the [MTSEA] to give a second-order rate constant of 485 ± 82 M−1s−1 (n = 13, biological replicates). (C) Same experiment as in A, except 0.2 mM MTSET was applied for L = 500 ms. Mean ± SEM for at least n = 3 (biological replicates)…

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  Figure 6.. Application of MTS reagents to the inactivated T449A/L472C, T449A/P473C//T449, and T449 Shaker-IR channels. The pictograms on the left indicate the status of the activation (lower) and inactivation (upper) gates. Inside-out patches were repeatedly depolarized from a holding potential of −120 to +50 mV to elicit K+ current through the channels. The timing and the duration of the MTS exposure (in ICS) to the inactivated channels are indicated by the hatched bars. Peak currents for each pulse (I(t)) were normalized to the peak current of the last pulse prior to MTS application (I0), averaged, and plotted as a function of cumulative modification time (t). The abscissa shows the cumulative exposure to MTS reagents throughout the figure. (A and B) 1-s-long depolarizing pulses were applied for T449A/L472C channels. MTS application was initiated 300 ms after the start of the depolarizing pulse (L = 500 ms). Normalized peak currents upon repeated treatment with 0.2 mM MTSET (green symbols) or 2 mM MTSEA (red symbols) are shown. (C and D) The modification of the inactivated T449A/P473C//T449 heteromeric channels was determined during repeated 3.0 s depolarizations to +50 mV. A decrease of…