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Figure 2. Gating currents of ShH4 W434F (A) and ShH4 Î6-46 W434F (B) (continuous lines) with the corresponding time integral (dashed lines). ON gating currents are identical at all potentials tested (here from â70 to 10 mV by 20-mV increments) between the two clones. For depolarizations below the opening threshold (â50 mV), the OFF gating currents are identical too, but for depolarizations leading to channel opening, ShH4 W434F OFF gating currents have a markedly slower decay than those of ShH4 Î6-46 W434F. HP = â90 mV, the pulse potential is indicated next to the traces. The external solution (top and guard) was NaMES Ca2; the internal solution (bottom) was K-Glu. The records are unsubtracted. (A) Data were sampled at 5 kHz and filtered at 1 kHz. The ON pulse duration was 50 ms. (B) Data were sampled at 2.5 kHz and filtered at 500 Hz. The ON pulse duration was 48 ms. A long OFF pulse (165 ms) allows recording of most of the charge return. Sweeps were made every 5 s to allow full recovery between traces.
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Figure 3. Chargeâvoltage relationship in ShH4 W434F channels. QON (â) and QOFF (â)measured from â120 to 6 mV in 2-mV increments. QON was best fitted to the sum of two Boltzmann distributions (Q1 = 5 nC, z1 = 2.9, V1(1/2) = â51.6 mV and Qâ2 = 27.3 nC, z2 = 5.6, V2(1/2) = â41.3 mV, where Q1 and Qâ2 are the amplitudes, V1(1/2) and V2(1/2) the half-activation potentials, and z1 and z2 the effective valences of the two distributions) and a straight line (slope 13.7 à 10â3 nC · mVâ1) to account for the linear capacity of the oocyte, which was compensated analogically as much as possible before recording, at a holding potential of â120 mV. The external solution was NaMES Ca2 and the bottom solution was K-Glu.
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Figure 4. OFF gating currents stepping back to â90 mV after a depolarization to â30 mV, for ShH4 W434F and ShH4 Î6-46 W434F. The traces are scaled to the peak. The initial time course (1) is identical for the two clones, but the second component has a much slower time course in the case of ShH4 W434F (3) than for ShH4 Î6-46 W434F (2), as one corresponds to a charge return from an inactivated state and the other from the open state.
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Figure 5. Quantification of two components in the OFF charge in ShH4 W434F. (A, top) OFF gating current returning to â90 mV after a 75-ms pulse to â30 mV. The slow component Qs was estimated by fitting the late part of the trace (36â165 ms after repolarization) starting from the cursor and extrapolated to time 0 of repolarization (shaded area). The fit was adequate for depolarizing pulses between â50 and 2 mV maintaining the same time constant for the slow component. The fast component Qf (bottom) was calculated by subtraction of this slow component (57 ms) from the total OFF gating current. For potentials more negative than â50 mV, we fitted only the fast component. (B) Qf, Qs, and QOFF total plot against the membrane potential. The superimposed lines are simultaneous fit of Qf and Qs to a single Boltzmann distribution (Qf max = â7 nC, Vf(1/2) = â59 mV, zf = 3.2, Qs max = â25.1 nC, Vs(1/2) = â40.7 mV, zs = 6, where Qf max and Qs max are the maximal amplitudes, Vf(1/2) and Vs(1/2) the half-activation potentials, and zf and zs the effective valences of the fast and slow components, respectively), and of QOFF total to the sum of these two Boltzmann distributions.
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Figure 6. Voltage dependence of the proportion of Qs and Qf in ShH4 W434F. (A) After a 100-ms pulse to 0 mV, the two components of the OFF gating currents were quantified for repolarizing potentials from â145 to â25 mV in 20-mV increments, as in Fig. 5. The total QOFF, as well as the slow (Qs) and fast (Qf) components amplitudes are plotted as a function of the repolarizing potential. (B) The ratio of Qf over the total charge QOFF as a function of the repolarizing potential has a maximum of â¼45% at â145 mV and a minimum of â¼12% at â80 mV.
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Figure 7. Absolute values of QOFF at different times after repolarization. (A) Unsubtracted records of OFF gating currents for 75-ms depolarizations to â40 (thin trace) and 0 mV (thick trace) from HP = â90 mV, shown along with the corresponding inverse of the time integrals (dotted lines). Though the charge moved at â40 mV is smaller than at 0 mV, the fraction of charge coming back in the first milliseconds is larger, as illustrated by the crossing of the charge integrals â¼8 ms after repolarization. (B) Absolute values of QOFF measured at different times after repolarization (as shown next to the data points). For very short times after repolarization, the chargeâpotential relationship has a bell shape rather than the usual Boltzmann distribution because the channels have to reach the open state (and inactivate) for the charge to be immobilized.
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Figure 8. Time course of charge immobilization. (A) Unsubtracted gating currents recordings for a depolarization to 0 mV of increasing duration (0.5â 23 ms in 1.5-ms increments), followed by a repolarization to â90 mV (HP = â90 mV). Data were sampled at 20 kHz and filtered at 4 kHz. (B) Evolution of the ratio of the fast returning charge over the total OFF charge as a function of the depolarizing pulse duration, fitted to a single exponential of time constant Ï = 4.4 ms for this cell. The time constant varies from 3 to 5 ms at 0 mV, as the one of ShH4 ionic currents inactivation at the same potential.
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Figure 9. ShH4 ionic current evoked by a 40-ms depolarizing pulse to 20 mV, just before and after 24 min of internal perfusion of an oocyte with NMDG-Glu (400 μl/h). The traces are scaled to the peak current, with amplitudes indicated next to the corresponding trace. In this cell, ionic current decayed with a time constant of 4.3 ms at the beginning of the experiment, and of 3.3 ms 24 min later, as the peak current was only 5% of the initial one. The perfusion pipette was placed close to the cytoplasmic face of the dome membrane. The current was monitored every 5 s, from a HP = â90 mV. The top and guard solution was NaMES Ca2, the bottom and perfusion pipette solution was NMDG-Glu. The data were sampled at 10 kHz and filtered at 2 kHz. A P/â4 protocol from SHP = â90 mV was used for leak subtraction.
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Figure 10. (A) Protocol used to evaluate the ionic and gating currents. A 50-ms prepulse to 20 mV was followed by a recovery pulse of variable duration (1â8,193 ms) and amplitude (â120, â90, â70, and â50 mV), and then by a 40-ms test pulse to 20 mV (HP = â90 mV). The records were made allowing 10 s between sweeps to avoid inactivation accumulation. The traces are unsubtracted. (B) The traces corresponding to recovery pulses of 1 and 8,193 ms are shown. The amount of recovery is estimated by the difference between the level of the prepulse plateau and the peak current during the test pulse. (C) Ionic currents after a recovery pulse to â120 mV of duration 1, 2, 3, 5, 9, 17, 33, 65, 129, 257, 513, 1,025, 2,049, 4,097, and 8,193 ms. The data were sampled at 10 kHz and filtered at 2 kHz.
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Figure 11. Comparison of the recovery of ionic current and gating charge at different potentials. Ionic currents and gating chargeâ normalized recoveries are superimposed for recovery potentials of â120, â90, â70, and â50 mV (n = 10 for ionic currents and 3 for gating currents; error bars = ±SEM). The overall time courses are close to the two types of currents for recovery at â120 or â90 mV. However, at more depolarized recovery potentials, the ShH4 ionic currents recover faster than ShH4 W434F gating currents.
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Figure 12. Voltage dependence of the recovery rates. (A) The rate of fast recovery of the ionic current (n = 13â15; ±SEM) and of the gating current (n = 4; ±SEM) are plotted as a function of the recovery potential. Each component was fitted to a rate that is an exponential function of the potential. The fitted zδ of ionic and gating current fast components are similar (0.39 and 0.49, respectively). The values of the rates are (sâ1): 67.4 ± 3.5, 47.7 ± 9.2, 28.6 ± 1.6, and 22.0 ± 1.2 for the ionic currents, and 124 ± 7.5, 83.7 ± 15.6, 43.9 ± 7.8, and 26.0 ± 4.28 for the gating currents (mean ± SEM). (B) The rate of slow recovery of the gating currents (n = 4, ±SEM) is plotted as a function of the recovery potential. The values of the rates are (sâ1): 26.6 ± 4.4, 16.9 ± 0.6, 7.11 ± 0.54, and 1.65 ± 0.15 (mean ± SEM). The data points cannot be fitted to a single exponential function of the potential. The two lines on the graph correspond to rates with zδ's of 0.4 and 1.7.
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Figure 13. Different kinetic schemes used to simulate ShH4 fast inactivation properties. Models 1A, 2, and 3 are derived from a sequential model developed for ShH4 Î6-46 and ShH4 Î6-46 W434F channels (Bezanilla et al., 1994; Roux et al., 1995). A flicker state Cf has been added, as described in Zagotta et al. (1994). The values of the parameters controlling the transitions involving inactivated states were determined through a visual fit to the experimental data. See Table I for the values of the parameters. Model 1B is the model proposed to account for ShB Î6-46 ionic current by Zagotta et al. (1994), with the addition of one fast inactivated state IN. The value of the θ parameter was set to 1, because our deactivation rate (ShH4 Î6-46 and ShH4) was much faster than the rates reported by Zagotta et al. (1994). The inactivation parameters values used were the same as for Model 1A (see Table I).
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Figure 14. (AâC) Ionic current traces predicted by Models 1A, 2, and 3, respectively, shown in Fig. 13. Traces are obtained from â90 mV holding potential; depolarizations are from â40 to 40 mV in 20-mV increments. (D) Ionic current traces recorded in the same conditions as simulated data shown in AâC (2 mM external K+, 120 mM internal K+, sampled at 10 kHz and filtered at 2 kHz). The traces were scaled to the peak current value at 40 mV.
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Figure 15. (AâC) Ionic current recovery predicted by Models 1A (A), 2 (B), and 3 (C) (solid lines), respectively, superimposed to the same data set of ionic current recovery (mean ± SEM) for recovery potentials of â120 (â¢), â90 (â), â70 (â´), and â50 (âµ) mV. Both simulation and experimental data correspond to recovery after a conditioning pulse of 50 ms to 20 mV. (D) The ratio of Qf over the total charge QOFF is plotted as a function of the repolarizing potential. After a 100-ms pulse to 0 mV, the membrane was repolarized to potentials between â150 and â70 mV, and the fast and slow component of charge return were quantified for the experimental data (â¢), for the linear sequential model (Model 1A) (â), for the two inactivated states model (Model 2) (âµ), and for the five inactivated states model (Model 3) (â´). Model 1A does not fit the experimental data, while Models 2 and 3 show a similar tendency as the experimental data.
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Figure 16. (AâD) OFF gating current evoked by repolarization to potentials from â150 to â70 mV in 10-mV increments, after a 20-ms depolarizing pulse to 20 mV from a holding potential of â90 mV, as predicted by Models 1A (A), 2 (B), and 3 (C), or as recorded experimentally (D). Predicted data and experimental data were generated/sampled at 10 kHz and filtered at 2 kHz. The data were normalized according to the amount of gating charge moving during the depolarizing pulse. (E and F) ON gating current evoked by depolarizations to potentials from â50 to 10 mV in 20-mV increments from a holding potential of â90 mV, as predicted by Model 3 (E) or as recorded (F). Data were generated/ sampled at 10 kHz, filtered at 2 kHz, and normalized to the peak gating current at 10 mV.
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