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Figure 1. Lack of inactivation in Sh-IR-T449V-I470C macroscopic current. Ionic currents elicited by pulses of 8 s from −90 mV to the indicated potential, in Sh-IR (left) and Sh-IR-T449V-I470C (right). Current records are acquired in the cell-attached patch configuration in symmetric K+ solutions. Note the lack of inactivation and the slow deactivation of the tail currents in Sh-IR-T449V- I470C.
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Figure 2. Single channel activity of Sh-IR and Sh-IR-T449V-I470C at +50 mV. Cell-attached patch recordings from oocytes expressing Sh-IR (A and B) and Sh-IR-T449V-I470C (C and D). From a holding potential of −90 mV, the membrane patches containing a single channel were depolarized to +50 mV for ∼80 s (the pulse protocol is shown above the records). The channel activity in Sh-IR persisted for ∼10 s before the channel fully inactivated (A). In Sh-IR-T449V-I470C (C), the channel activity was sustained throughout the depolarization and persisted for a few seconds after repolarization to −90 mV. The activity of both channels is also shown in an expanded time scale (B and D). Sh-IR was sampled at 10 kHz and filtered at 2 kHz; T449V-I470C was sampled at 5 kHz and filtered at 1 kHz. Long records are shown decimated. (E) Single channel IV plot for Sh-IR(•) and T449V-I470C (○) in symmetrical 120 mM K+. The chord conductance calculated for Sh-IR and T449V-I470C was 17 and 9 pS, respectively.
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Figure 3. Single channel activity of Sh-IR and Sh-IR-T449V-I470C at −45 mV. (A) Consecutive recordings in a cell-attached configuration (120 mM K+ in bath and patch pipet) from an oocyte expressing Sh-IR-T449V-I470C. The membrane patch contained a single channel. At least three patterns of opening are present: brief openings (Mode 1, *); sustained activity characterized by a higher open probability with some flickering (Mode 2, **); and long openings without flickering (Mode 3, ***). The ensemble average of 30 records from the same patch is shown in B. (C) Single channel activity in Sh-IR at −45 mV; only brief openings are present. The patch contained at least two channels. (D) Ensemble average of 40 records from a patch containing at least four channels, same oocyte as in C. The pulse protocol is shown in the top panels; arrows indicate closed (c) and the open (o) states.
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Figure 4. Kinetics and current-voltage relationships are practically indistinguishable in Sh-IR-T449V-I470C and Sh-IR during short depolarizations. K+ current families from oocytes expressing (A) the double mutant Sh-IR-T449V-I470C and (C) Sh-IR evoked by 40 ms pulses ranging from −80 mV to + 50mV in 5-mV increments, from a holding potential of −90 mV. The corresponding IV curves are shown in B and D.
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Figure 5. In Sh-IR-T449V-I470C and in Sh-IR, long depolarizations produce a change in the voltage dependence of charge movement. Gating currents recorded in NMG-MES solution from K+-depleted oocytes using the cut-open oocyte voltage clamp technique. (A) Gating currents from Sh-IR-T449V-I470C recorded from −90-mV holding potential during voltage steps from −120 to 20 mV in 10-mV increments (SHP = −120 mV; P = −4). (B) Gating currents from the same oocyte recorded from 0-mV holding potential and during hyperpolarizations ranging from −10 to −170 mV in −10-mV increments (SHP= 0 mV, P = −6). The plot in C shows the time integral of the ON gating currents (QV curve) for the two holding potentials (HP = 0 mV, •, and HP = −90 mV, ○). D and E show unsubtracted gating currents from Sh-IR in identical conditions as in A and B. The corresponding QV curves showed in F illustrate the shift to more negative potentials of the QV from slow inactivated channels (HP = 0 mV, •, HP = −90 mV, ○).
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Figure 7. Two components in the tail current of Sh-IR-T449V-I470C. After conditioning depolarization to 0 mV for 0.01, 0.04, 0.07, 0.1, 0.2, 0.4, 1.0, and 2.0 s, the decaying phase of the tail currents at −90 mV were simultaneously fit to the sum of two exponential functions with the constraint that the two time constants were maintained in all traces. (A) Shows tail currents recorded after 10-, 200-, and 2,000-ms pulses to 0 mV with the respective fit superimposed. The fast and slow time constants common to all tail currents were 1.1 and 141.4 ms, respectively. The plot in B shows that the amplitude of the fast component (τ = 1.1 ms) remained practically unchanged with the different pulse duration. On the other hand, the amplitude of the slow component (τ = 141.4 ms; C) increased with the duration of the depolarization, whereas it was practically undetectable for the shortest depolarization (10 ms).
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Figure 8. Tail currents during repolarizations at different potentials. Top panels show superimposed tail current records during repolarization to the indicated potentials, elicited after 50- or 1,000-ms pulse to 0 mV. In Sh-IR (A), the deactivation kinetics are practically identical after long (1,000 ms) or short (50 ms) depolarizations. (B) Same type of experiment as in A, but from an oocyte expressing Sh-IR-T449V-I470C. After 1,000-ms depolarizations to 0 mV, tail currents are significantly slower. (C–F) Sh-IR-T449V-I470C: superimposed deactivation tail currents at different repolarizing potentials after prepulses of 50 and 1,000 ms. Note that the voltage shift required to obtain similar time courses of deactivation tails after 50- and 1,000-ms pulses is not linear, but increases for more negative potentials.
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Figure 6. The deactivation rate of Sh-IR-T449V-I470C depends on the duration of depolarizations. The top panel shows K+ tail currents from an oocyte expressing Sh-IR during repolarization to −90 mV. The tail currents are recorded after depolarizations to 0 mV of increasing duration (the duration of the preceding pulse to 0 mV is shown next to the traces). Note that the duration of the conditioning depolarization does not affect the rate of deactivation. In the bottom panel, the same experiment as in A but from an oocyte expressing Sh-IR-T449V-I470C. The tail currents show the appearance of a slow component that progressively increases with pulse duration.
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Figure 9. Correlation between changes in the voltage dependence of charge movement and the increase of the slow component of the tail current in Sh-IR-T449V-I470C. (A) Gating currents evoked by a voltage step from 0 mV to −60 mV recorded from a K+-depleted oocyte (see pulse protocol in top panel). The oocyte was held at −90 mV, depolarized to 0 mV for different times (as indicated next to current traces) before the pulse to −60 mV. As shown, the gating currents were progressively reduced as the depolarization time at 0 mV increased because of the change in the voltage dependence of the charge movement. The reduction in the charge movement as a function of the duration of the depolarization is plotted in B. C shows the increase in the slow component of tail current as a function of duration of depolarization to 0 mV. Data points in B and C were fitted with single exponential functions with time constants of 557ms (B, dashed line) and 440 ms (C, dashed line). Both data sets in B and C could also be well fitted simultaneously by exponential functions with identical time constant of 459 ms (continuous lines).
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Figure 10. Single channel properties during deactivation in Sh-IR. The records (cell attached patch configuration) show the single channel activity at −90 mV sampled after a depolarization to 0 mV for 50 ms (A) and for 1,000 ms (B). The pulse protocol is shown above the records. Note that during repolarization, the channel activity ceased rapidly when the membrane potential returns to −90 mV for short and long pulses. Currents were sampled at 5 kHz and filtered at 1kHz.
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Figure 11. Single channel properties during deactivation in Sh-IR-T449V-I470C. The records (cell-attached patch configuration containing a single channel) show the single channel activity at −90 mV after a depolarization to 0 mV for 50 (A) and for 1,000 ms (B). Note that prolonged depolarizations stabilized a conducting state of the channel (B) that remained open for a relatively long time at −90 mV. Currents were sampled at 5 kHz and filtered at 1kHz.
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Figure 12. Variance-mean plot in Sh-IR (A) and Sh-IR-T449V-I470C (B). Variance-mean plots taken from an ensemble (n = 201) of current traces recorded in cell attached patch configuration and evoked by 40-ms depolarization to 30 mV, followed by repolarization to −100 mV. Depolarization and repolarization data were fit to the equation: Variance = iI − I2/N, where i represents the single channel current amplitude, I is the mean current, and N is the number of channels; g is the conductance of the single channels. (A and B) are the results for Sh-IR and Sh-IR-T449V-I470C, respectively. The parameters used for the fitting were as follows. Sh-IR at −100 mV, g = 24.0, and POmax 90.2%; at +30 mV, g = 15.8, POmax = 87.3%, and N (at −100 and +30) = 3,538. For Sh-IR-T449V-I470C at −100 mV, g = 8.4, POmax = 42.2%, and N = 2,694; at +30mV, g = 7.1 POmax = 67.0%, and N = 1,380. N was estimated independently for −100 and 30 mV in Sh-IR-T449V-I470C.
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Figure 13. A second open state of Sh-IR-T449V-I470C revealed by conditional open probability. A–D are COP (P(t2|t1)) plots computed from ensemble of ionic tail currents using to the equation P(t2|t1) = C(t1,t2)/iI(t1) + I(t2)/Ni, where C(t2|t1) is the autocovariance function, I(t) is the mean current, and “i” and “N” are obtained from the mean-variance analysis shown in Fig. 12. The conditional open probability at −100 mV at reference times t1 = 0.5 and 8 ms, after a 40- ms depolarization to 30 mV, is shown for Sh-IR (A) and for Sh-IR-T449V-I470C (B). In both clones, the decays of the COP after short depolarizations (A and B) were well fitted by a single exponential function (continuous line). The time constants of the COP decay are reported in the plot. The decay of COP at −100 mV after long depolarization (300 ms) are shown in C for Sh-IR and in D for Sh-IR-T449V-I470C. Data in C and D are fitted to a sum of two exponential functions. The results of the fitting are shown in the panels. Note that, even after long depolarizations, the COP in Sh-IR decays practically monoexponentially (C) for both reference times. On the contrary, the COP in Sh-IR-T449V-I470C decays biexponentially (D). The plots in E and F show the relationship between the time constant of the COP decay with the reference time. In Sh-IR-T449V-I470C, after short depolarizing pulses, the time constant of conditional open probability decay increases quickly as a function of t1 and pulse duration (F), an indication of the presence of more than one open state. In Sh-IR (E), the time constant of the decay is practically independent from the reference time chosen.
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Figure 14. A model for Sh-IR-T449V-I470C. The model is adapted from Olcese et al. 1997, and describes equilibrium properties of slow inactivation in Sh-IR. The rate constants used for the activation pathway were identical to those in Bezanilla et al. 1994. The rate constants leading to and away from the open state (O) are, for the potential −100 mV (in ms−1): α = 11.33, β = 5.23. The voltage independent rate constants of inactivation (γ = 0.00026 ms−1, δ = 4.22 × 10−6 ms−1) and the interaction energy (ln ω = −2.427 kT) are identical to the model of Olcese et al. 1997, except for the additional binding factor θ = 0.01, which was added to slow transitions out of the Io state. (A) Simulated COP decay of tail currents (V = −100 mV) for the case of nonconducting inactivated state (INC), and that of conducting inactivated state (IC), after short depolarizations to 30 mV (20 ms). Two reference times are shown (see arrows) at 0.2 and 20 ms. Data were obtained from 380 groups of 5,000 accumulated runs filtered at 5 kHz, and the COP was computed according to . (B) COP decay after a long depolarization (1 s). Data acquisition is the same as in A, except for filter rate (500 Hz). Reference times (arrows) are 2 and 30 ms. Insets show ionic current for the two models, which are similar for short depolarizations, but diverge for long depolarizations.
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