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Figure 2. Tail current kinetics change as C-type inactivation develops (A and B). (A) Recordings obtained in symmetric (115 mM) Na+ solutions, using test pulses (to +40 mV) of differing durations demonstrate that development of C-type inactivation parallels significant changes in tail current waveform. After short depolarizations, they are dominated by a rapid component associated with deactivation of noninactivated channels, followed by a small slow ionic component. This slow ionic current component increases with increasing depolarization duration. Finally, a rising phase (hook) in the onset of the slow ionic current becomes apparent while the initial, fast peak disappears as C-type inactivation approaches steady state. (B) Recordings obtained under ionic conditions where the estimated reversal potential for Na+ (115 mM versus 38 mM) nearly matches the potential of depolarization (+20 mV) such that IgON becomes clearly visible. Upon increasing duration of the depolarizing pulse segment, the tail currents undergo kinetic changes similar to those seen in A. Data traces were low pass filtered at 4 kHz.
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Figure 3. Dependence of inward tail current waveforms on the development of C-type inactivation, without (A and B) and with (C and D) addition of external Na+ ions. Inward currents were measured in the absence of intracellular permeating ions (Tris-EGTA in the bath), during pulses to â20 mV in external 2.5 mM K+ plus 115 mM Tris (A and B) or in NFR containing 2.5 mM K+ plus 115 mM Na+ (C and D). In these solutions, channels inactivate with a time constant of â¼1 s. In the absence of Na+ (A), the tail currents upon repolarization to â120 mV are rapid and decrease in magnitude as the degree of inactivation increases (see B). In the presence of external Na+ (C), however, tail currents are only rapid after very brief depolarizations. After longer depolarizations (i.e., as C-type inactivation occurs), tail currents slow down and do not decrease in size. D shows the tail current sections of C, aligned in time to the end of the test depolarization. Note the 10-fold change in time scale compared with B. E shows an analysis of data from C and D, demonstrating a linear relationship between the fraction of channels that have entered into C-type inactivation and the peak magnitude of the slow component of Na+ tail currents.
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Figure 4. Shaker channels become less permeable to K+ ions and relatively more permeable to Na+ ions when C-type inactivated. (A) Superposition of four current traces recorded in NFR versus Na-EGTA; i.e., in symmetrical 115 mM Na+ but with 2.5 mM K+ added to the external solution. Pairs of traces were recorded with a depolarization to â20 mV for 200 ms and 3 s leading to quite different degrees of inactivation at the end of the depolarization. Subsequently, the potential was stepped to either â40 or +40 mV. After the short depolarization, the subsequent step to +40 mV shows only marginal outward current, the same step after almost complete inactivation results in outward currents that are â¼50% of the size of the inward currents obtained at â40 mV, indicating an enhanced relative Na+ permeation after development of C-type inactivation. (B) Demonstration of the protocol used for collection of instantaneous I-V data at two different durations within a â20-mV test depolarization. Ramp potential changes of 40-ms duration were imposed after 20-ms (B, a and b) and 10.04-s (B, c and d) test pulses. Ramps were upward (from â20 to +70 mV) in B, a and c, and downward (from +70 to â20 mV) in B, b and d. See text for further description of this protocol. (C) Reversal potentials obtained from instantaneous I-V data are plotted as functions of the degree of C-type inactivation that had occurred before starting the ramps. Results are shown for eight patches from which full data sets were collected. Straight lines connect data points from the same patch. Upward ramp data points are shown by open symbols and connected by solid lines, downward ramp points are shown by filled symbols and connected by dashed lines.
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Figure 5. Influence of intracellular K+ on the tail currents. The first trace (i) was recorded with external Li-Ringer (same results would be obtained with Na+ instead of Li+ as permeating ion, not shown) and an internal solution containing 5 mM K+ plus 110 mM Tris+. The arrow points to an inflection in the tail currents indicating two components. The following traces (iiâiv) were recorded while the internal K+ ions were slowly washed out by Tris-EGTA solution. As the outward currents disappear, due to removal of internal K+ ions, the tail currents become slower and larger, indicating increased Li+ permeation.
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Figure 6. Deactivation of C-type inactivated channels. (A and B) Tail currents were recorded after depolarizations to +20 mV for 100 ms at tail potentials between â170 and â80 mV using 10-mV steps. Representative traces are shown in A and B using different time scales to demonstrate the voltage sensitivity of deactivation in A and of the hooked rising phase in B. The recording bandwidth was 4 kHz. The time constants of both, the hooks (ÏCd1) and the deactivation (ÏCd2), were estimated by fitting double-exponential functions to the data. The mean time constants resulting from six experiments are plotted in C as a function of the tail potential. The filled symbols indicate those data points that were considered for the determination of the slopes (straight lines) that yielded a voltage dependence for both processes corresponding to effective gating valences of 0.51 (ÏCd1) and 1.05 e0 (ÏCd2).
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Figure 7. Activation of ionic current through C-type inactivated channels. Recovery from C-type inactivation is affected by external cations (A). Double-pulse experiments were performed in the indicated solutions. The duration of the interpulse interval at â100 mV was chosen such that the slow tail currents in Na/Na solutions reach the baseline. In Na/Na solutions (top) depolarization after complete tail current deactivation shows only partial recovery from inactivation. In Tris/Na solutions (bottom), no significant recovery from inactivation is visible. Instead, slow, mono-exponential reactivation to the same steady state level as in the first pulse is observed (dashed lines). (B) Activation of current through C-type inactivated channels is shown in Tris/Na solutions in response to depolarizations from â100 mV to voltages between â30 and +80 mV in steps of 10 mV. The depolarizations were given in rapid succession such that no marked recovery from inactivation occurred (see A). The activation kinetics (under these conditions of steady state inactivation) were estimated by fitting single-exponentials functions to the data. The resulting time constants are plotted in C as a function of the test potential. The straight line corresponds to a single exponential function in this semilogarithmic plot. The slope of this line yields an estimate for the apparent gating charge associated with the process of reactivation of 1.13 e0.
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Figure 8. Effects of internal K+ ions on recovery from C-type inactivation. Currents were recorded in the absence of extracellular permeating ions (Tris-Ringer), individual traces being separated by 15-s intervals. Between A, B, and C, bath solutions were altered as indicated. In A, in the presence of 1 mM internal K+, channels undergo rapid and complete inactivation (A, i and ii). Switching to K+-free 115 mM internal Na+ solutions results in a similarly fast inactivation (B, i) that falls to a steady state value that indicates continued Na+ permeation through C-type inactivated channels (see also Fig. 7 A). The next pulse in Na+-solution (B, ii) shows no recovery from inactivation, thus this trace shows only activation of C-type inactivated channels (as in Fig. 7 B). Switching back to K+-containing internal solutions reveals almost complete inactivation (C, i). However, the final pulse (C, ii) shows partial recovery of ânormalâ inactivation.
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