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Figure 1. . Rate of gating charge recovery in wild-type and W434F mutant channels. The rate of charge recovery was assessed by using a double pulse protocol. A pair of pulses to 0 mV was applied to inside-out patches separated by varying durations at â100 mV. Both the external and internal solutions contained 1 mM K+ + 115 mM Tris+ to achieve a reversal potential of 0 mV. (A) The depolarizing pulse to 0 mV evokes an ON gating current, then large inward tail currents are seen through the wild-type channels during the repolarizing pulse, and the ON gating current in the second pulse recovers with increasing interpulse durations. (B) Only the OFF gating current is present in the nonconducting mutant during repolarization. (C) Direct comparison of the rate of tail current decay and that of charge recovery in the wild-type channel indicates that channel closing is fast compared with charge return. The actual normalized tail current trace and the fit to the recovering second ON gating currents from A are shown. (D) Charge recovery is significantly faster in the wild-type channels (tau = 2.5 ms for this patch) than in the W434F mutant (tau = 5.9 ms for this patch). The fits were taken from A and B and are shown normalized for direct comparison.
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Figure 2. . Relationship between gating charge recovery and channel closure in the wild-type and the W434F channel. The percentage of gating charge recovery was determined using the double pulse protocol by calculating the ratio of the ON gating charge in the second pulse to that in the first pulse (left). Tail currents from the left panels are shown on the middle panels on a different time scale. Solutions used in the experiments were (A) symmetrical 1 K+ + 115 Tris+ for wild-type and (B) symmetrical 115 Na+ for W434F channels. (A) In the wild-type channel the relationship between charge recovery and channel closing is nonlinear in 1 K+ solutions, (B) but it is linear in the W434F mutant in 115 Na+ solutions. In the wild-type, 13% of the charge recovered (A, left) while the tail current dropped 48% compared with its peak value (A, middle) in this patch; however, (B) in W434F a tail current loss of 54% was accompanied by 58% charge recovery. These results from many patches (WT: n = 29; W434F: n = 18) and various interpulse durations are summarized in the point plots (right).
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Figure 3. . Charge recovery and tail current time constants. (A, top) In the wild-type channel, charge recovery rates were similar in ionic conditions that produced different tail current decay rates (B, top). (A and B, middle) Different segments of the same three traces recorded during a double pulse protocol illustrate the similar amounts of gating charge recovery but different rates of tail current decay during the 3-ms interpulse interval in the three ionic conditions. The segments shown correspond to the solid line on the pulse pattern (bottom). The peak gating currents in the first depolarizing pulse were used to normalize the traces in A, and tail currents were normalized by their peaks in B. (C, top) In contrast to the wild-type channel, charge recovery time constants show a strong dependence on the ionic species in the W434F mutant. (C, middle) Recovery of gating currents in the indicated solutions from different patches is sensitive to ion species. Symmetrical solutions were used, and labels indicate the main cation and its concentration in mM. In 1 mM K+ and 1 mM Rb+ solutions, the substitute ion was 115 mM Tris+. Bars indicate the mean ± SEM of single exponential time constants determined for individual patches. The number of patches was as follows, wild-type: 115 Cs+, n = 16; 1 Rb+, n = 22; 1 K+, n = 29. W434F: 115 Cs+, n = 8; 115 Rb+, n = 10; 115 K+, n = 15.
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Figure 4. . Low permeation and slow channel closing in W434F. (A and B) The external side of an outside-out patch was exposed to solutions containing 115 mM K+, Cs+, Rb+, or Tris+, whereas the pipette solution (internal solution) contained 115 mM Tris+. Traces on A and B are from the same patch and are only separated for clarity. Currents were elicited by a 15-ms step to 0 mV from a holding potential of â100 mV, followed by a step back to â100 mV. For external Tris+, only the ON and OFF gating currents are seen during depolarizing and repolarizing steps, respectively. By contrast, small inward currents are also seen during the depolarizing step in the other external solutions, which increase to noticeable tail currents during the repolarizing step. (C) The ratio of the integrated charge in the OFF gating + tail current to that in the ON gating current indicates significant permeation only with 115 K+ in the external solution (QOFF/QON = 2.82 ± 0.27, n = 8). (D) The decay of the tail current was significantly slower in K+ than in other conditions. In other external ions, tail decay rate consistently showed the following sequence: Cs+ > Tris+ > Rb+, but these differences were not statistically significant. (E) The relationship between gating charge recovery and channel closure in the mutant channel is linear in 115 K+O// 115 Tris+I solutions. The figure was prepared as described in the legend to Fig. 2.
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Figure 5. . The relationship between gating charge recovery and channel closure in the wild-type channel in 115 Na+O//115 Na+I solutions. The figure was prepared as described in the legend to Fig. 2. (A) Na+ tail currents have at least two components in the wild-type channel after short (<60â100 ms) first depolarizing pulses. After a 15-ms depolarizing pulse â¼30% of the tail current decays with the fast component (middle). Due to this, charge recovery lags behind channel closing, which is illustrated by the point plot (right, n = 14). (D) After a 1,000-ms depolarizing pulse the fast component of the tail current disappears and the relationship between gating charge recovery and tail current loss becomes 1:1 linear (right, n = 13), like it is in the W434F mutant. The data traces shown were obtained from different patches. Percentage values indicate the fraction of recovered gating charge on the left panels and the fraction of tail current amplitude decrease on the middle panels for these recordings.
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Figure 6. . The slow component of charge return becomes apparent in scatter plots. The fraction of recovered gating charge is plotted as a function of the interpulse duration used in the double pulse protocol. A single exponential fit to all data points yields a time constant of 2.4 ms, but this function gives a poor fit for interpulse durations over 5 ms (inset, dashed line). A double exponential fit with fast and slow time constants of 1.8 and 28 ms and relative weights of 85 and 15%, respectively, fits the data points well (solid line in both figure and inset). This scatter plot contains data recorded in symmetrical 1 K+ + 115 Tris+ solutions from 29 patches containing wild-type channels.
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Figure 7. . Fast time constants of charge recovery and their relative weights in wild-type and mutant channels. Data were obtained from double exponential fits to scatter plots in the indicated ionic conditions. Symmetrical solutions were used and the labels indicate the main cation and its concentration in mM in both solutions. When a concentration of 1 mM was used for an ion, it was complemented by 115 mM Tris+. For most conditions, the slow time constant fell in the 18â30 ms range. When in some conditions the double exponential fit yielded unreasonably slow time constants, it was constrained within this range to get meaningful values. This did not affect the quality of the fit. For this reason the slow time constants are not shown in the figure. For the wild-type channel, the values in parentheses for the 115 Na+ condition indicate the duration of the first depolarizing pulse in the double pulse protocol.
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Figure 8. . The effect of external barium on charge recovery rates in the W434F mutant. (A) The overall charge recovery rates are comparisons based on single exponential fits in external solutions containing 1.8 mM Ca2+ (dark bars) and external solutions containing 2 mM Ba2+ (light bars). Labels indicate the composition of the external//internal solutions. Overall, barium had a stronger effect on the rate of charge return in 115 Na+ + 2.5 K+O//115 K+I solutions than in symmetrical 115 Na+ solutions, and had no effect in symmetrical 115 Tris+ solutions. Bars indicate the mean ± SEM of single exponential time constants determined for individual patches. (B and C) Fast time constants of charge recovery and their relative weights obtained from double exponential fits to scatter plots, as described in the legend to Fig. 7. Barium decreased the weight of the slow component in every condition, most notably in Na+O//Na+I. Barium accelerated the fast component when K+ was present, but did not affect it in Na+ and Tris+ solutions.
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Figure 9. . High affinity block of W434F channels by barium in Na+ and Tris+ solutions. (A) Micromolar concentration of barium blocks W434F channels in K+-free solutions. Depolarizing steps of 20 ms duration to 60 mV were applied every second from a holding potential of â100 mV. Currents were recorded from an outside-out patch containing W434F channels. The external solution contained (in mM) 36 Na+ + 79 Tris+, and the internal solution was 115 Na+, yielding a reversal potential of about â30 mV. Switching to an external solution containing 1 μM Ba2+ resulted in an almost complete block of both the outward current and the inward tail current in five pulses. (B) Inward dissociation of Ba2+ is slow in a Tris+ internal solution. Recordings are from an outside-out patch containing W434F channels. The pipette solution (internal solution) contained 115 Tris+. The external solution originally was 115 Na+. A step pulse to 0 mV for 15 ms followed by a return to â100 mV evoked a small inward Na+ current followed by a large Na+ tail current (trace 1). The external solution was then switched to 115 Tris+ + 2 Ba2+, which abolished all ionic currents, leaving only the gating currents (trace 2). After switching back to 115 Na+ again, the first trace recorded 8 s after the switch showed very little tail current (trace 3), indicating that most of the channels were still blocked by Ba2+. Further washing resulted in the full recovery of the Na+ current (trace 4) with a time constant of 38 s.
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Figure 10. . Barium affects the rate of charge recovery from both the deep and the external lock-in sites. The traces shown were recorded in symmetrical 115 Na+ solutions from an outside-out patch containing W434F channels. From a holding potential of â100 mV the voltage was stepped to 0 mV for 15 ms, then returned to â100 mV for 5 ms, and then returned again to 0 mV. (A) In the presence of 10 mM external Ba2+ charge recovers quickly, which is indicated by the fast decay of the OFF gating current (Ï = 3 ms) and the high fraction of recovered charge in the second ON pulse (73%). The QOFF/QON2 ratio, which is the ratio of integrated charge in the OFF gating current + tail current to that in the ON gating current in the second pulse, shows that there is no inward Na+ permeation during the repolarizing pulse. (B) 2 s after switching to Ba2+-free solution the QOFF/QON2 ratio is still close to unity, signaling negligible permeation, but there is a significant slowing in the OFF gating current decay (Ï = 5 ms), which is also reflected in the lower fraction of recovered charge in the second ON gating pulse (61%). This suggests that Ba2+ has left from the external lock-in site, but not the deep site. (C) 46 s after the switch a large Na+ tail current is seen during the repolarizing pulse (QOFF/QON2 = 9.34), signaling Ba2+ exit from the deep site, and further slowing of the charge recovery rate is observed (Ï = 11 ms, Qrec = 54%).
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Figure 11. . The effect of internal cations on OFF gating current kinetics. Inside-out patches containing W434F channels were exposed to internal solutions containing 115 mM Tris+, Na+, K+, Rb+, or Cs+, whereas the external solution was NFR + 5 mM Ba2+. Under these conditions, the occupancy of the deep site by Ba2+ is high and barium's interaction with the ion in the cavity will determine the rate of channel closing and OFF gating current decay. Channels were opened by a 15-ms pulse to 0 mV then the voltage was returned to the holding potential of â100 mV eliciting the OFF gating currents. Each part of the figure shows results from a different patch. (A) OFF gating current decay was the slowest with Tris+ as the internal cation (Ï = 5.62 ms in this patch), was faster in Na+ (Ï = 1.25 ms), and still faster in K+ (Ï = 615 μs). (BâD) Differences in OFF gating kinetics were small or negligible when the internal solution was switched between any two of K+, Cs+, or Rb+. B, switch between Cs+ (Ï = 394 μs) and K+ (465 μs); C, between Rb+ (477 μs) and K+ (564 μs); and D, between Cs+ (434 μs) and Rb+ (407 μs).
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Figure 12. . Block of K+ conduction in the wild-type channel by external Ba2+ slows the rate of charge recovery. The traces were recorded from an inside-out patch in symmetrical 1 K+ + 115 Tris+ solutions with the addition of 2 mM Ba2+ in the pipette. From a holding potential of â100 mV a pair of pulses to 0 mV was applied, separated by 2 ms at â100 mV. Trace 1, recorded first, shows some fast inward K+ tail current and a greater amount of charge recovered in the second pulse (56%). As barium in the pipette solution diffuses into the channels, it blocks the potassium tail current, leaving only the OFF gating current (trace 2). Along with the disappearance of the tail current, a clear slowing of the charge recovery rate is visible in the second ON gating current (32%).
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Figure 13. . W434F channels are slow to enter the C-inactivated state. Traces were recorded in symmetrical 115 Na+ solutions from an inside-out patch containing W434F channels. The standard double pulse protocol was used with (A) a 15-ms and (B) a 10-s first pulse duration. Despite the dramatic increase in the duration of the first pulse, little slowing in charge recovery is seen (15 ms first pulse: 83%, 10 s first pulse: 71%). The interpulse duration was 15 ms. The large Na+ tail currents were omitted from the figure for clarity.
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Figure 14. . A model to account for the effects of cations on the gating charge recovery in wild-type and W434F channels. In accordance with current models of activation, the channel has to traverse a series of closed states before opening. The transition into I0 is modulated by cations from the external lock-in site, being the fastest in Na+ and Tris+. A fraction of the channels move on to C-inactivated states that are associated with the shift of the Q-V function. Reaching deeper C-states results in progressively slower charge recovery. Upon repolarization, wild-type channels close quickly from the O state, but the ones recovering from I0 close more slowly into closed-inactivated states through R â CIn due to the slower intrinsic rate of this transition. In the mutant channel, the closing rate of the gate is the same in both branches, and is similar to the closing rate of the wild-type in the lower branch. Closing of the gate in both the top and bottom branches is prevented by ions occupying the cavity, which is represented by the O+ion and R+ion states. Inward ion permeation or internal Tris+ causes a higher population of the O+ion and R+ion states and thus hinders channel closing. The slow recovery from the C-inactivated states generates the slow component of charge recovery. The fast I0 â R transition along with the higher Na+ conductance of the R state is responsible for the rising phase of Na+ tail currents in both channel types.
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Figure 15. . The three components of Na+ tail currents in the wild-type channel. The trace was recorded in 115 Na+O//115 Na+I solutions from an inside out patch containing wild-type channels. The tail current was elicited by stepping back to â100 mV after a 15-ms long depolarizing pulse to 0 mV. The original data trace is shown with the three exponential components of the fit. The short dashed line indicates the fast component (Ï = 236 μs, channels closing from the open state), the long dashed indicates the intermediate component (Ï = 4.3 ms, channels returning from I0), and the solid line indicates the slow component (Ï = 25.5 ms, channels returning from IC1).
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