Article Figures & Data
Figures
- Fig. 1.
Initial phase of recovery in 150 mmexternal K+. A, Examination of the speed of voltage change and steady-state voltage control in two-electrode voltage clamp of an oocyte. The voltage change is 62% (of total) at 0.1 msec, 83% at 0.2 msec, 95% at 0.3 msec, and ∼99% at 0.4 msec.B, The oocyte was held at −80 mV and pulsed twice to +30 mV (each for 60 msec) every 3 sec. The intervening gap between the two pulses is set at various potentials (recovery potential,Vr) and lengthened by 0.2 msec between each sweep. The currents in the second pulse are used as a measure of the extent of recovery from inactivation produced by the first pulse. No matter thatVr is −70 mV or −220 mV (inset is an enlarged redrawing of the currents for Vr −70 mV), the currents in the second pulse appear within 0.2 msec. The initial rate of recovery, however, is faster at more negative Vr. Thesolid lines denote zero current level, and thedashed lines mark the sustained (noninactivating) current level. C, Time courses of recovery in the first ∼3 msec for Vr −70 mV to −250 mV by the same experimental protocol as that in B. Thehorizontal axis is the length of Vr. Thevertical axis is the extent of recovery (fraction recovered), which is the difference between the peak current of the second pulse and the current at the end of first pulse, divided by the difference between the peak current of the first pulse and the current at the end of the first pulse. The first three to five points at each potential are fit by linear regression functions (dashed lines) of the form: fraction recovered = slope ·t (t denotes length of Vrin msec), in which slope equals 0.060 (−70 mV), 0.085 (−100 mV), 0.12 (−130 mV), 0.17 (−160 mV), 0.25 (−190 mV), 0.33 (−220 mV), and 0.43 (−250 mV). D, The common logarithms of the slopes of the regression lines in C are plotted againstVr. The solid line is a linear regression fit of the form: y = −1.52 − 0.0047x, indicating that the initial recovery rate increases e-fold per ∼90 mV [(loge)/0.0047 = 92] of hyperpolarization.
- Fig. 2.
Extended time course of recovery in 150 mm external K+. A, Time courses of recovery at Vr −40, −70, and −100 mV. Voltage protocols and plots are as in Figure 1 except that the length ofVr is much longer here. Smooth curves are monoexponential fits of the form: fraction recovered = 1 − exp(−t/τ) (t denotes length ofVr, the horizontal axis), in which τ equals 27.2, 19.0, and 12.7 msec for the data in −40, −70, and −100 mV, respectively. B, Time courses are similar to those inA but are at Vr −130, −160, and −190 mV, with the time course at Vr −100 mV inA plotted here again for comparison. Note that the time courses “cross” each other. The smooth curves are two-exponential fits of the form: fraction recovered = 1 − 0.64 · exp(−t/5.3) − 0.36 · exp(−t/32) (−130 mV), = 1 − 0.61 · exp(−t/3.2) − 0.39 · exp(−t/44) (−160 mV), and = 1 − 0.55 · exp(−t/1.9) − 0.45 · exp(−t/38) (−190 mV). C, The fast time constants from the two-exponential fits in four cells are 5.6 ± 0.4 msec (−130 mV), 3.4 ± 0.3 msec (−160 mV), and 2.1 ± 0.3 msec (−190 mV). D, The slow time constants from the same data pool in C are apparently voltage-independent and are 39 ± 6 msec (−130 mV), 42 ± 2 msec (−160 mV), and 40 ± 3 msec (−190 mV). E, The proportion (preexponential factor) of the slow component from the same data pool in C increases with increasing hyperpolarization (0.31 ± 0.05 at −130 mV, 0.36 ± 0.03 at −160 mV, and 0.46 ± 0.02 at −190 mV).
- Fig. 3.
The slow inward tail currents at various repolarizing potentials in 150 mm external K+.A, The oocyte is held at −80 mV and pulsed every 3 sec to +60 mV for 30 msec and then repolarized to various potentials for 45 msec to demonstrate the slow inward tail currents. B, The decaying time constants of the slow inward tail currents from four cells are 6.1 ± 0.6 msec (−130 mV), 3.4 ± 0.2 msec (−160 mV), and 1.9 ± 0.2 msec (−190 mV), which correlate well with the fast time constants of macroscopic recovery (the same data as those in Fig. 2C) at various repolarizing potentials.
- Fig. 4.
Initial phase of recovery in 150 mmexternal Na+. A, Time courses are similar to Figure 1C on the basis of the same experimental protocols. The first six points at each potential are fit by linear regression functions (dashed lines) of the form: fraction recovered = slope · t, in which slope equals 0.013 (−70 mV), 0.019 (−100 mV), 0.026 (−130 mV), 0.037 (−160 mV), 0.052 (−190 mV), 0.07 (−220 mV), and 0.09 (−250 mV).B, The common logarithms of the slopes of the regression lines in A are plotted against Vr. Thesolid line is a linear regression fit of the form:y = −2.19 − 0.0047x.C, The y-intercepts of regression lines similar to that in B in various external solutions are −2.21 ± 0.25 (150 mm external Na+, 5 cells), −1.55 ± 0.04 (150 mm external K+, 4 cells), −1.51 ± 0.11 (300 mmexternal K+, 3 cells), and −1.57 ± 0.03 (500 mm external K+, 3 cells). D, The slopes of the regression lines in C are 0.0045 ± 0.0004 (150 mm Na+), 0.0047 ± 0.0002 (150 mm K+), 0.0046 ± 0.0005 (300 mm K+), and 0.0046 ± 0.0001 (500 mm K+).
- Fig. 5.
Extended time courses of macroscopic recovery in 150 mm external Na+. A, Time courses of recovery at recovery potential (Vr) −100, −160, and −220 mV. Voltage protocols and plots are as in Figure 2. The time courses also “cross” each other, yet the recovery at 100 msec becomes incomplete, especially at more negativeVr. The smooth curves are mono- or two-exponential fits of the form: fraction recovered = 0.95 − 0.95 · exp(−t/37) (−100 mV), = 0.92 − 0.35 · exp(−t/8.6) −0.57 · exp(−t/53) (−160 mV), and = 0.82 − 0.22 · exp(−t/3.3) − 0.60 · exp(−t/39) (−220 mV). B, The fast time constants from the two-exponential fits in three cells are 8.0 ± 0.7 msec (−160 mV), 4.8 ± 0.9 msec (−190 mV), and 3.0 ± 0.5 msec (−220 mV).
- Fig. 6.
Recovery time course at extremely negative recovery potential (Vr) in 300 mm external K+. Voltage protocols and plots are as in Figure 2. AtVr −130 mV, the recovery essentially is complete at 100 msec, whereas at Vr −250 mV the recovery is far from complete at 100 msec. The smooth curves are two-exponential fits of the form: fraction recovered = 1 − 0.75 · exp(−t/5.1) − 0.25 · exp(−t/41) (−130 mV), and = 0.81 − 0.63 · exp(−t/0.9) − 0.18 · exp(−t/42) (−250 mV).
- Fig. 7.
An elaboration of scheme 2 summarizing the findings. +V and −V mean that a reaction process is accelerated with depolarization and hyperpolarization, respectively. Because the unbinding rates of the inactivating particle are smaller from the more deactivated channel, the binding rates onto the more deactivated channel also must be smaller so that channel inactivation is still coupled to activation. The longitudinal arrows, therefore, are made smaller toward theleft side of the scheme. The rate constants of some key steps in the scheme are (all in msec−1): unbinding rate from the OB state, α (in 150 mm external K+) = 0.028 · exp(Vm/−90) or α (in 150 mmexternal Na+) = 0.006 · exp(Vm/−90), in which Vm denotes membrane potential; deactivation rate of the OB state, γ = 0.003 · exp(Vm/−47); deactivation rate from the O state, δ > 2 at −100 mV and even faster at more negative potentials. These numbers mostly are derived directly from the experimental findings, except that γ is a postulated function to well recapitulate the observed proportions of fast and slow components of recovery. The unbinding rate from the CB state (CB to C rate) may be set at a voltage-independent value ∼0.025 msec−1, because the slow time constants of the recovery course are always ∼40 msec at −130 to −190 mV. Such voltage independence, in contrast to the definite voltage dependence of α, implies that the activation–deactivation conformational changes alter the electrical lines across the pore; thus, the bound inactivating particle could no longer sense the electrical field in the closed channel.
