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Figure 2. Ionic currents of Shaker wild-type, NH2 terminus truncated monomer (ShÎ) and tetrameric constructs recorded with the two-microelectrode voltage clamp. (A) WWWW currents; (B) FWWW currents; (C) FWFW currents; (D) ShÎ currents; (E) peak G-V curves for the various channel types; (F) steady-state inactivation; the fitted curves are Boltzmann functions with midpoint voltages of â20, â23, â37, and â48 mV, and effective valences of 2.6, 2.6, 4.2, and 3.6 for ShÎ, WWWW, FWWW and FWFW, respectively. In each part of the figure, the holding potential was â90 mV, the bath solution was ND 96, and, except for the data plotted in F, leak currents were subtracted by the P/4 protocol (Bezanilla and Armstrong, 1977) with â120 mV leak holding potential. Depolarizing test potentials were from â50 to +30 mV in 20-mV steps in A, B, C, and D. Conductance in E was computed assuming a linear open-channel current-voltage relationship and a reversal potential of â85 mV. For the experiments in F, prepulses ranged from â150 to +30 mV in 30-mV steps and were 10 s in duration. They were followed by +30-mV depolarizing test pulse for 5 s; pulse sequences were delivered every 40 s, and leak current was subtracted using single P/4 pulses from a â140 mV leak holding potential.
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Figure 3. Effect of extracellular potassium on inactivation kinetics and peak currents of the three tetrameric constructs. (A) Normalized current time courses. Test pulses to +40 mV, 8-s duration were applied from a holding potential of â90 mV. The 2 mM K+ bath solution was ND 96; the given K+ concentration for the other solutions was obtained by replacing Na+ with K+. (B) Time constants of one (WWWW) or two (FWWW, FWFW) exponentials fitted to the inactivation time course at +40 mV. For FWWW the amplitude of the faster component was 73â82% of the total relaxation; for FWFW it ranged from 30 to 73% for the curves shown. (C) Relative peak currents at +40 mV in the various solutions normalized to the current in 2 mM K+. Error bars in B and C represent standard deviations with n = 6 in each case.
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Figure 4. Effect of monovalent cations on inactivation kinetics and peak currents. (A) Normalized currents. Holding potential was â90 mV, test potential was +40 mV for 8 s. Cation concentrations are as indicated; 96Na refers to ND96 solution which contains 2 mM K+. In the other solutions the indicated cation replaced both Na+ and K+. (B) Time constants from single or double-exponential fits to the decay time courses. (C) Relative peak currents in the various solutions normalized by current in the 96Na solution.
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Figure 5. TEA effects on inactivation and activation. (A) Effect of 30 mM TEA in the bath solution (replacing Na+ in ND96) on wild-type tetramer currents. Depolarizations to +30 mV were given for 50 s from a holding potential of â90 mV. The dissociation constant for channel block estimated under these conditions was 30 ± 7 mM (n = 14). (B) Currents from FWWW tetramers. External TEA slows the time course of inactivation. (C) FWFW tetramer currents. Inset shows the initial currents on an expanded time scale. (D) Voltage dependence of normalized peak conductance of FWFW in the absence and presence of 30 mM TEA. (E) Voltage dependence of FWFW inactivation in the absence and presence of 30 mM TEA. Prepulses of 10-s duration were followed by a +30-mV test pulse; the pulse sequence (including a single P/4 pattern) was repeated at 40-s intervals. From fits of Boltzmann functions the midpoint voltage and effective valence were â51 mV and 3.6 for FWFW currents in ND96, â51 mV and 3.2 in 30 TEA. (F) Time course of recovery from inactivation at â90 mV in the standard ND-96 solution or in external solutions containing 100 mM K+ or 30 mM TEA. Plotted for FWFW is the peak current of a second depolarization to +30 mV, relative to that from an initial 5-s depolarization to +30 mV. For WWWW currents, which are incompletely inactivated by the 5-s prepulse, the fractional recovery (Levy and Deutsch, 1996) is plotted. Mean values for six experiments are shown in each case. âRecovery timeâ is the interval at â90 mV between the two depolarizations. Pulses were delivered at 60-s intervals.
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Figure 6. Inside-out patch recordings from FWFW-injected oocytes with the standard solutions (141 mM K+ in the bath, 5 mM K+ in the pipette). (A) Seven successive sweeps in a one-channel patch, showing bursts of openings in response to depolarization to +30 mV from â90 mV holding potential. Data were filtered at 2 kHz. The ensemble mean time course, obtained from 200 sweeps, shows a rapid decay from the maximum open probability of 0.3. The superimposed smooth curve is a single exponential with Ï = 10 ms. (B) Seven successive sweeps in a different one-channel patch show long bursts of openings in response to depolarizations to +30 mV from â90 mV holding potential. Filter bandwidth was 1 kHz. The ensemble mean time course, obtained from 100 sweeps, shows slow decay from an open probability of 0.6. The superimposed curve is a single exponential with Ï = 150 ms. (C) FWFW macroscopic current from a patch recording at +30 mV, filtered at 2 kHz. The superimposed smooth curve is the sum of two exponentials with time constants of 10 and 150 ms; the amplitudes of the components were 153 and 47 pA, respectively. (D) Time constants obtained in unconstrained, two-exponential fits to macroscopic currents at various potentials; same FWFW patch as in C. For single-channel recordings in A and B, leak subtraction used the average of null sweeps.
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Scheme I.
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Figure 7. Activity of an FWFW single channel. (A) Diary plot showing the integrated open time in each of 220 depolarizations to +30 mV, 400-ms duration, delivered at 5-s intervals from a holding potential of â90 mV. Same patch as in Fig. 6 A. Of a total of 219 sweeps, 71 showed activity; the 69 runs of active and inactive sweeps is significantly (P < 0.002) smaller than the 97 runs expected for random activity. (B) Histogram of times in the âavailableâ state. The mean dwell time was 1.9 sweeps; the superimposed curve represents a geometric distribution with this mean value. (C) Histogram of times in the âunavailableâ state. The mean dwell time was 3.8 sweeps.
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Figure 8. Single channel conductance of ShÎ, WWWW, and FWFW channels in the standard solutions. (A) Representative current amplitude histograms at +30 mV. For accumulation into all-points histograms, data were filtered at 2 kHz for ShÎ and WWWW, 5 kHz for FWFW. (B) Single channel current as a function of voltage obtained from double-Gaussian fits to amplitude histograms. Fitted lines have slopes of 13, 12, and 13 pS for ShÎ, WWWW, and FWFW respectively.
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Figure 9. Open and closed time histograms from ShÎ, WWWW, and FWFW single-channel recordings. (A) Three successive sweeps from a one-channel recording of ShÎ and the corresponding open and closed time histograms. (B) WWWW currents. (C) FWFW currents. Data were filtered at 1.4 kHz for display and analysis in A and B, 2 kHz in C. Histograms contain more than 11,000 entries for ShÎ and WWWW, 630 entries for FWFW. Solid curves show maximum-likelihood fits of one exponential (Open times) or the mixture of two exponential distributions (Closed times).
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Figure 10. Cell-attached patch recording of W434F channels with 140 mM K+ pipette solution. (A) Mean current (predominantly gating current) in response to depolarization to +80 mV from â80 mV holding potential; the average of 100 sweeps is shown. The patch contained 2,400 channels, as estimated from the integrated gating current assuming 13 e0 of charge movement per channel. P/5 leak subtraction was used with a leak holding potential of â120 mV. (Contamination of gating current during the P/5 pulse produces the artifact preceding the âoffâ current.) Data were filtered at 5 kHz. (B) Six of the 164 sweeps with detectable ionic currents, selected from a total of 1,400 sweeps recorded. Data were filtered at 3 kHz and are displayed after subtraction of the mean of 100 sweeps to remove gating and leak currents. (C) Time course of open probability estimated from idealization of the sweeps containing channel activity. The superimposed curve is an exponential function with 1 ms time constant. The pipette solution contained (in mM) 140 K-aspartate, 1.8 CaCl2, 10 HEPES, pH 7.4.
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