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Volume 17, Number 10,
Issue of May 15, 1997
pp. 3436-3444
Copyright ©1997 Society for Neuroscience
Deactivation Retards Recovery from Inactivation in Shaker
K+ Channels
Chung-Chin Kuo
Department of Physiology, National Taiwan University College of
Medicine, and Department of Neurology, National Taiwan University
Hospital, Taipei 100, Taiwan, Republic of China
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
In Na+ channels, recovery from inactivation
begins with a delay, followed by an exponential course, and
hyperpolarization shortens the delay as well as hastens the entire
exponential phase. These findings have been taken to indicate that
Na+ channels must deactivate to recover from inactivation,
and deactivation facilitates the unbinding of the inactivating
particle. In contrast, it is demonstrated in this study that recovery
from inactivation in Shaker K+ channels begins with no
delay on repolarization. Moreover, hyperpolarization hastens only the
initial phase (fast component) of recovery yet retards the later phases
of recovery by increasing the proportion of slow components. The time
course of slow inward "tail" K+ currents, which
presumably result from the open state(s) traversed by the recovering
inactivated channel, always matches the fast, but not the slow,
components of recovery, suggesting that the fast and the slow
components primarily correspond to recovery via the open state
(unblocking of the inactivating particle before channel deactivation)
and via the closed state (deactivation before unblocking),
respectively. Besides, changing external K+ concentration
effectively alters the absolute value of the initial recovery speed,
but not its voltage dependence. It seems that Shaker K+
channel deactivation hinders, rather than facilitates, the unbinding of
the inactivating particle and therefore retards recovery from inactivation, whereas external K+ may enhance unbinding of
the inactivating particle by binding to a site located near the
external entrance of the pore.
Key words:
two-electrode voltage clamp;
Shaker K+
channel;
deactivation;
ball and chain model of inactivation;
recovery
from inactivation;
K+ ion binding site
INTRODUCTION
K+ channels are important
proteins controlling membrane excitability. Different K+
channels may show different gating kinetics, which are crucial in
considering the role of the channels in shaping cellular firing patterns (Rudy, 1988 ; Hille, 1992). The fast inactivation of Shaker K+ channels is termed N-type inactivation, to be
distinguished from the other, slower inactivation (Iverson and Rudy,
1990; Hoshi et al., 1991). Like in Na+ channels, the
development of N-type inactivation is explained by the "ball and
chain" model (Armstrong and Bezanilla, 1977; Armstrong, 1981).
According to the model, inactivation is produced by blocking the open
channel with an inactivating particle (a peptide "ball"), which
probably corresponds to the N-terminal region of the Shaker
K+ channel protein (Hoshi et al., 1990; Zagotta et al.,
1990)
scheme 1
[View Larger Version of this Image (9K GIF file)]
Above is a simplified gating scheme incorporating the foregoing
concepts of fast inactivation, in which OB and CB denote open and
closed conformations blocked by the inactivating particle, respectively. Basically, the route C to O to OB should be the major or
even exclusive pathway for the development of inactivation so that
inactivation is "coupled" to activation, and most channels would
not be inactivated (blocked) until being "used" (open). On the
other hand, the inactivated channel (state OB) in principle may recover
via the OB to O to C (unblocking before deactivation) route or the OB
to CB to C (deactivation before unblocking) route, and the two routes
have very different physiological meanings. The former implies
substantial ionic currents through the channels traversing state O
during recovery, whereas the latter assures no such "leak" or
"slow tail" currents on repolarization. It is interesting that
Na+ channels and Shaker K+ channels, although
sharing similar molecular steps in the development of fast
inactivation, seem to behave differently in recovering from
inactivation. In Na+ channels the "deactivation before
unblocking" route seems to be exclusively preferred to assure
negligible leak Na+ currents during recovery (Kuo and Bean,
1994). On the other hand, many inactivated Shaker K+
channels seem to take the OB to O to C route to recover because there
are substantial macroscopic K+ currents or single channel
openings on repolarization (Demo and Yellen, 1991; Ruppersberg et al.,
1991).
The OB to O to C route, however, may not be the exclusive pathway of
recovery for inactivated Shaker K+ channels. Demo and
Yellen (1991) found that 8% recovery is via the closed state in 160 mM external K+ at  80 mV, and the percentage
is higher at 120 mV or in 30 mM external K+,
suggesting modulation of the recovery processes by membrane potentials
and external K+. Because different recovery pathways imply
different physiological consequences, it is desirable to explore the
mechanisms modulating the molecular operations of recovery in Shaker
K+ channels in more detail. It is demonstrated herein that
the modulatory effects of external K+ and membrane
potentials could be understood by kinetic interactions within a
simplified gating scheme and that deactivation of the inactivated
Shaker K+ channel probably retards, rather than enhances,
the exit of the bound inactivating particle.
MATERIALS AND METHODS
Molecular biology and oocyte injection. All
experiments were performed with mRNA synthesized from the Shaker GH4
K+ channel cDNA, which is essentially the same as Shaker H4
channel reported by Kamb et al. (1987), but with the addition of three "silent" restriction enzyme sites for BglII,
BstII, and SmaI, as well as a larger 5
untranslated region. Plasmids containing the ShGH4 cDNA were linearized
by digestion with the appropriate restriction enzymes. Then linearized
template cDNA was used for the synthesis of mRNA by standard methods,
using T7 polymerase (MacKinnon and Yellen, 1990). Oocytes were
harvested from mature Xenopus laevis females
previously injected with human chorionic gonadotropin and dissociated
in calcium-free OR-2 solution [(in mM) 82.5 NaCl, 2.5 KCl,
1 MgCl2, and 5 HEPES] with collagenase (Type IA, 1-2
mg/ml; Sigma, St. Louis, MO) for 1-2 hr. Isolated, follicle-free stage
V to stage VI oocytes were selected and injected with 50 nl of
synthetic mRNA in distilled water (0.1-1 mg/ml). Injected oocytes were
maintained at 18°C in ND-96 solution [(in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES plus 50 mg/ml gentamycin] until subsequent recordings. With daily change of the ND-96-maintaining solution, the injected oocytes usually survived for at least 7-10 d.
Current recording and data analysis. Whole-cell Shaker
K+ channel currents from the injected oocytes were usually
of decent size and suitable for recording 2-3 d after injection. For
recordings, oocytes were transferred to a chamber with continuous
superperfusion of 150 mM K+ [(in
mM) 150 KCl, 1 CaCl2, 1 MgCl2, and
10 HEPES, pH 7.4] or 150 mM Na+ or 300 or 500 mM K+ solutions. The composition of the latter
three solutions is the same as that of the 150 mM
K+ solution, except that 150 mM KCl is replaced
by 150 mM NaCl or 300 or 500 mM KCl,
respectively. The 300 and 500 mM K+ solutions
are so hypertonic that the oocyte usually must go through one or two
solutions of intermediate osmolarity (created by adding sucrose to
ND-96) before being transferred into these recording solutions.
Currents were measured at room temperature (~22°C) by a standard
two-electrode voltage-clamp amplifier (Warner OC-725B-HV; Hamden, CT),
supported by a 12 bit analog/digital converter controlled by a
laboratory personal computer. Data were sampled at 5-10 kHz and
filtered at 2 kHz (8-pole Bessel filter). Linear, digital leak
subtraction was performed off-line using leak currents measured by a
hyperpolarizing pulse from 80 to 90 mV. Statistics are given as
mean ± SD.
RESULTS
The recovery from inactivation in Shaker K+ channels
begins with no delay in 150 mM external K+
In Na+ channels at the beginning of recovery there is
a delay ascribable to the deactivation process (OB to CB in scheme 1) that the channel must go through before the inactivating particle could
unblock (Kuo and Bean, 1994). If the inactivated Shaker K+
channel may recover via the OB to O to C route, then no intermediate step is needed for the inactivating particle to unblock, and the recovery therefore should begin with no delay. Figure 1
characterizes this very initial phase of recovery in Shaker
K+ channels. Figure 1A monitors the
voltage change of an oocyte in the two-electrode voltage clamp, showing
faithful voltage control with satisfactory "clamp speed." The
voltage change is typically >60% in 0.1 msec and essentially is
completed after 0.3 msec. Figure 1B illustrates that
at either 70 mV or 220 mV there is always unequivocal recovery
during the first 0.2 msec of repolarization. This is consistent with
previous reports (Demo and Yellen, 1991; Ruppersberg et al., 1991) and
suggests that in 150 mM external K+ many
inactivated Shaker K+ channels take the OB to O route to
recover from inactivation. Because the rate of development of C-type
inactivation in wild-type Shaker H4 channels is ~1-2
sec 1 in 10-100 mM external K+
(Baukrowitz and Yellen, 1995), all inactivating pulses used in this
study have been kept short (30-60 msec) to avoid significant contamination from other, slower inactivation process than N-type inactivation. This also is checked by examining the current of those
Shaker GH4 channels for which the N-type inactivation is removed by
deleting amino acid residues 6-46 (in 10-100 mM external K+ the rate of development of C-type inactivation in these
mutated channels is not very different from that in wild-type channels; see Baukrowitz and Yellen, 1995). It is found that the currents generally decrease by no more than 3-5% over such short depolarizing pulses (see also Heginbotham and MacKinnon, 1993) (data not shown). Thus C-type or other, slower inactivation probably could be
disregarded, and the consideration would be focused on N-type
inactivation.
Fig. 1.
Initial phase of recovery in 150 mM
external 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 that
Vr 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. The
solid lines denote zero current level, and the
dashed 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. The
horizontal axis is the length of Vr. The
vertical 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 Vr
in 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 against
Vr. 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 [(log
e)/0.0047 = 92] of hyperpolarization.
[View Larger Version of this Image (24K GIF file)]
The initial recovery rates increase consistently with increasing
hyperpolarization in 150 mM external K+
Figure 1C depicts the time courses of recovery in the
first ~3 msec of repolarization. The recovery begins with no delay
and follows an initial linear course, the slope of which is larger at
more negative potentials. Figure 1D plots a common
logarithm of the slope against membrane potential, with a regression
line to indicate that the initial recovery rate consistently increases e-fold per ~90 mV of hyperpolarization between 70 and
250 mV. Another interesting finding in Figure 1C is that
the time course at 250 mV appears to "bend" more than the course
at 190 mV when the recovery fraction exceeds ~0.3. Thus the time
courses at 190 mV to 250 mV are clearly apart at the beginning yet
are converging at ~3 msec. This implies that hyperpolarization
probably accelerates only the early phase, but not the late phase, of
recovery.
Strong hyperpolarization increases the proportion of a late slow
recovery phase
The effect of hyperpolarization on the late recovery phase
is investigated by examining the complete time courses of recovery in
Figure 2. Figure 2A shows that the
recovery courses at 40 to 100 mV are all essentially completed
within 100 msec and can be approximated by monoexponential functions.
At 130 mV or more negative potentials the recovery courses can be
described only by two-exponential functions and start to "cross"
each other (Fig. 2B), clearly indicating that
increasing hyperpolarization hastens the initial phase, but not the
late phase, of recovery. The fast time constants of these
two-exponential recovery courses become smaller with increasing
hyperpolarization (approximately e-fold change per 60 mV,
Fig. 2C). On the other hand, the slow time constants of
these courses seem to be voltage-independent (Fig.
2D), although the proportion of the slow components
increases as the membrane potential gets more negative (Fig.
2E).
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 of
Vr is much longer here. Smooth curves are
monoexponential fits of the form: fraction recovered = 1 exp(t/ ) (t denotes length of
Vr, the horizontal axis), in which 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 in
A but are at Vr 130, 160, and 190
mV, with the time course at Vr 100 mV in
A 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).
[View Larger Version of this Image (26K GIF file)]
The slow component of recovery represents recovery via the
deactivated states
Because increasing hyperpolarization hastens the entire
course of recovery in Na+ channels (Kuo and Bean, 1994), it
is very interesting to see that stronger hyperpolarization hastens only
the immediate recovery from inactivation yet drives more inactivated
channels into some other states from which recovery is actually
retarded. Based on scheme 1, a most straightforward explanation for
these findings would be that increasing hyperpolarization not only
speeds the unblocking of the inactivating particle from the
open-blocked channel (OB to O or the initial recovery rate) but also
enhances deactivation of the open-blocked channels (OB to CB). The
essentially unchanged time constants of the slow component of recovery
in Figure 2D then would suggest that CB to C is the
slower (rate-limiting) step of the OB to CB to C route and possibly is
not voltage-dependent. It has been shown that the recovery from
inactivation in Shaker K+ channels is accompanied by
prominent inward "slow" tail K+ current in 150 mM external K+ (Demo and Yellen, 1991;
Gomez-Lagunas and Armstrong, 1994), which presumably results from the
traversed open state during recovery (route OB to O to C in scheme 1).
If the slow component primarily corresponds to recovery via the OB to
CB to C route, then it should not be accompanied by significant tail
currents. Figure 3A examines the slow tail
currents, which match the fast components of macroscopic recovery in
time course at various potentials (Fig. 3B) and clearly contain no "slow" phase corresponding to the slow component of recovery. This finding lends strong support for the view that the slow
component of recovery primarily results from those inactivated channels
recovering via the OB to CB to C route.
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.
[View Larger Version of this Image (13K GIF file)]
The initial recovery rates in 150 mM external
Na+ are remarkably slower but carry the same
voltage dependence
The recovery from inactivation was reexamined over the same wide
range of repolarizing potentials in an external solution containing 150 mM Na+, but no K+. Strictly
speaking, this is not a condition of "zero" but only "low"
external K+, because with the two-pulse protocol used here
the K+ ions flowing out during the first pulse may not be
dissipated fully before the repolarizing pulse (the local residual
K+ concentration is possibly 10-15 mM when
checked by the reversal potential of the deactivating tail currents
that follow a short depolarization pulse; data not shown). Similar to
the findings in 150 mM external K+, the initial
recovery phase in 150 mM Na+ (Fig.
4A) follows a linear course, the slope
of which, however, is consistently four to five times smaller than the
slope at the same repolarizing potential in Figure 1C.
Figure 4B plots a common logarithm of the slopes in
Figure 4A against membrane potential, with a
regression line having the same slope as, but a y-intercept 0.67 smaller than, the regression line in Figure 1D.
This indicates that in 150 mM external Na+ the
initial recovery rates also increase e-fold per ~90 mV of hyperpolarization yet are, in general, 100.67 or ~4.7
times slower than those in 150 mM K+ at every
potential tested. The same experiments also are performed in 300 and
500 mM external K+. The y-intercepts
of the regression lines similar to those in Figures
1D and 4B on average differ by 0.66 between 150 mM external Na+ and 150 mM external K+ but show no difference among
150, 300, and 500 mM external K+ (Fig.
4C). The slopes of these regression lines, on the other hand, always remain the same in either very low or very high external K+ (Fig. 4D).
Fig. 4.
Initial phase of recovery in 150 mM
external 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. The
solid 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 mM
external 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+).
[View Larger Version of this Image (21K GIF file)]
There is a very slow component of macroscopic recovery in low
external K+
Figure 5A shows more extended time
courses of recovery up to 100 msec in 150 mM external
Na+. The time courses also cross each other like those in
Figure 2B. At 160 mV or more negative potentials,
the time courses can be described only by two-exponential functions,
and the fast time constants are smaller as the membrane potential goes
more negative (Fig. 5B). For the late phase of recovery, it
is noteworthy that in 150 mM external Na+ the
recovery at 100 msec is clearly not complete at these negative potentials. This phenomenon is more pronounced at more negative potentials (Fig. 5A) as if, other than the fast and slow
components, there are some "very slow" components of recovery.
Although C-type or other slow inactivation may become more prevalent in
low external K+ (Baukrowitz and Yellen, 1995), it is hard
to envisage how these very slow components, if resulting from C-type
inactivation, should become more pronounced at more negative
repolarizing potentials. An alternative to explain this phenomenon is
to make scheme 1 less oversimplified by adding more closed states (see
scheme 2 below, in which V denotes those
hyperpolarization-accelerated key processes under discussion here).
Because the initial recovery rate (OB to O rate) is quite slower in low
external K+ than in high external K+ (Figs.
1C, 4A), the chance of taking the OB to CB
route, and consequently the chance of reaching state CB, would be
higher in low K+. With scheme 2 the very slow component of
recovery may be explained by a hypothesis that some inactivated
channels are moved into the CB state, along with a very slow
unblocking rate from the more completely deactivated state (very slow
CB to C rate).
scheme 2
[View Larger Version of this Image (8K GIF file)]
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 negative
Vr. 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).
[View Larger Version of this Image (14K GIF file)]
The very slow component of recovery also is observed in high
external K+ at extremely negative potentials
Considering that increased hyperpolarization would increase the
chance for a channel in state OB to recover via the OB to CB route
(Fig. 2E), if the very slow component of macroscopic recovery in low external K+ is indeed a result of simple
kinetic interactions rather than an effect of external K+
on some slow inactivation processes, then the very slow components of
recovery should become manifest even in high external K+ at
very negative recovery potentials. Figure 6 illustrates
that in 300 mM external K+ the recovery
essentially is complete after 100 msec at 130 mV, whereas at 250 mV
a very slow component (~20% of total) of macroscopic recovery
similar to that observed in Figure 5 occurs, and the recovery clearly
is incomplete after the same 100 msec period.
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. At
Vr 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).
[View Larger Version of this Image (12K GIF file)]
DISCUSSION
Quantitative analysis and support of the gating scheme
In this study the fast or initial phase of recovery from
inactivation is characterized in several different ways, namely the linear regression for the initial recovery rates, the exponential fit
for extended recovery time courses, and the decay of the slow inward
tail currents during recovery. The relationship among these measurements may be derived analytically on the basis of a more elaborated form of scheme 2 (Fig. 7). Focusing on the
fast recovery (OB to CB to C) route, we have:
|
(1)
|
in which OB(0) denotes the fraction of OB at the beginning of
repolarization, and t denotes time. The reverse reactions C to O and CB to OB are assumed negligible at the hyperpolarized recovery
potentials. The reaction O to OB also is neglected, because most
channels in state O would be "absorbed" to state C because of the
very rapid  (>2 msec1 at 100 mV, measured by the
decay of the deactivating tail currents after a short activating pulse;
data not shown) and the slow O to OB rate (~0.1 msec1,
measured by the decay of the macroscopic outward currents at +60 mV).
Solve the equations for O(t) and C(t):
|
(2)
|
C(t) + O(t) here is the extent of the fast
component of recovery. If the initial recovery rate (Fig.
1C) represents the OB to O rate ( ), it should be equal to
the product of the inverse of the fast time constant (+ ) and the
proportion of the fast component of the extended recovery time course
[/(+)]. This seems true by comparing such products from the
time courses at 70 to 190 mV in Figure 2 and the initial recovery
rates at the same potentials in Figure 1C. Furthermore, the
slow inward tail current at repolarization represents the proportion of
state O as a function of time, O(t). Because is quite
faster than (+), according to the above equation O(t)
would approximate a monoexponential decaying function (after a
transient initial rising phase) with a time constant of 1/(+),
the foregoing fast time constant. These are just the findings in Figure
3.
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 the
left side of the scheme. The rate constants of some key
steps in the scheme are (all in msec1): unbinding rate
from the OB state, (in 150 mM external K+) = 0.028 · exp(Vm/90) or (in 150 mM
external Na+) = 0.006 · exp(Vm/90), in
which Vm denotes membrane potential; deactivation rate
of the OB state, = 0.003 · exp(Vm/47);
deactivation rate from the O state, > 2 at 100 mV and even
faster at more negative potentials. These numbers mostly are derived
directly from the experimental findings, except that 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
msec1, 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 ,
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.
[View Larger Version of this Image (10K GIF file)]
Some other findings also may be discussed in the light of the same
quantitative arguments. For example, the initial recovery rates ()
are accelerated consistently e-fold per 90 mV
hyper-polarization between 70 and 250 mV (Figs.
1D, 4B), whereas the voltage
dependence of the fast time constants [1/(+)] seems stronger
(e-fold change per ~60 mV between 130 and 190 mV or
between 160 and 220 mV; Figs. 2C, 5B).
Moreover, the initial recovery rates in 150 mM external
K+ and in 150 mM external Na+
always differ by ~4.5 times at potentials between 70 and 250 mV
(Fig. 4C), but the fast time constants of macroscopic
recovery in these two conditions differ by only ~2.3 times (at 160
to 190 mV, Figs. 2C, 5B). With the foregoing
equations these findings may imply that is more voltage-dependent
than , and external K+ probably accelerates only ,
but not .
The route of recovery from inactivation for Shaker
K+ channels
Unlike Na+ channels, Shaker K+ channels
may take either the OB to O to C or OB to CB to C route to recover from
inactivation. With reactions O to OB and CB to OB being negligible
during recovery (see above), the preponderance of each route would be
determined by the ratio between and in Figure 7. In 150 mM external K+, 31-46% of recovery is via the
OB to CB route at 130 to 190 mV (Fig. 2E). These
are fairly close to the /(+) ratios in Figure 7 (29-43%) in
the same conditions. In low external K+, decreases, and
the chance of getting into the OB to CB route is consequently higher.
Figure 5A indicates that the fast components comprise only
35-23% of total recovery at 160 mV to 220 mV, or 65-77% of
total recovery is via the OB to CB route. These are again close to the
/(+) ratios in Figure 7 (72-82%). It is interesting to note
that the /(+) ratio is ~50% at 70 mV in low external
K+. Thus both recovery routes are probably similarly
important in the physiological condition.
Deactivation retards recovery from inactivation in Shaker
K+ channels: contrast with Na+ channels
Hyperpolarization apparently has two distinct effects on recovery
of the inactivated Shaker channels. It hastens the initial recovery
phase, presumably by facilitating the unblocking step OB to O, yet
retards the late recovery phase, presumably by forcing deactivation of
the inactivated channels. A very interesting connotation from this
finding is that the unblocking rate CB to C is significantly slower
than OB to O. The very slow components of recovery in Figures 5A and 6 further suggest that the unblocking rate is even
slower as the channel is deactivated more fully (very slow CB to C rate). This is as if the inactivating particle is "locked" in the
pore when the channel is forced to deactivate before the inactivating particle unblocks. The blocking of delayed rectifier K+
channels in squid giant axon by tetraethylammonium (TEA) ion derivatives has been shown to be similar to K+ channel
inactivation in many aspects, including the findings that the
unblocking of TEA derivatives is also four to five times faster when
external K+ is increased from 10 to 440 mM
(Armstrong, 1971) and that TEA binding also strongly immobilizes the
gating charges (Bezanilla et al., 1991; Perozo et al., 1993). In this
light, it is interesting to note that the recovery from the block by
TEA derivatives at 100 mV also shows a faster initial phase, yet a
slower late phase, as compared with the recovery at 60 mV (Armstrong,
1971).
Such a lock-in effect of the inactivating particle in deactivated
Shaker channels is in sharp contrast to the case in Na+
channels, in which stronger hyperpolarization monotonously speeds up
the whole course of recovery (Kuo and Bean, 1994). It seems that in
Na+ channels inactivation is coupled to activation, because
the receptor conformation for the inactivating particle is
"created" with channel activation and thus channel deactivation
would "eject" the inactivating particle by destroying the receptor
("modulated receptor"), whereas the activation/deactivation
processes in Shaker K+ channels alter the "doorway" of
the receptor ("guarded receptor"), with or without an as drastic
change of the receptor conformation itself.
Location of the external K+ binding site of which
the effective occupancy accelerates recovery from inactivation
Consistent with previous reports (Demo and Yellen, 1991;
Gomez-Lagunas and Armstrong, 1994), the initial recovery rates in 150 mM external K+ are four to five times faster
than those in 150 mM external Na+. The initial
recovery rate may be expressed in terms of the occupancy of an
enhancement site by external K+ (OP) as:
Note that when external K+ is exerting this
recovery-enhancing effect the pore is blocked by the inactivating
particle, so any K+ from the outside must go back to the
outside. Thus OP will change with the membrane potential according to
the electric distance of the site (Woodhull, 1973). Because different
OP at different potentials will contribute to the overall change of
initial recovery rate with voltage, the apparent voltage dependence of
the initial recovery rate may be larger than the intrinsic voltage
dependence of the unblocking rate (presumably the same in both enhanced
and unenhanced conditions) itself.
The same initial recovery rates in 150-500 mM external
K+ (Fig. 4C,D) suggest that the enhancement site
is nearly fully occupied in 150 mM (or higher) external
K+ at 70 to 250 mV (see also Murrell-Lagnado and
Aldrich, 1993). In other words, OP remains close to 1 and does not
change significantly with membrane potential in these conditions. The
observed voltage dependence of the initial recovery rates
(e-fold change per ~90 mV) therefore must be fully
ascribed to the intrinsic voltage dependence of the unblocking step. On
the other hand, the enhancement site seems far from fully occupied in
150 mM external Na+, because the initial
recovery rates here are four to five times slower. These initial
recovery rates, however, still change e-fold per 90 mV, the
intrinsic voltage dependence of the unblocking step. Thus OP does not
change significantly from 70 to 250 mV even when the site is by far
not saturated, indicating that the electric distance of the enhancement
site is negligible from outside. This K+ binding site thus
may be located near the external entrance of the K+ channel
pore, just like the high-affinity Ca2+ binding sites in the
L-type Ca2+ channel pore (Kuo and Hess, 1993) or even on
the external surface of the Shaker K+ channel. In either
case the enhancement phenomenon possibly represents an allosteric
rather than a direct "knock-off" effect, because the binding site
for the inactivating particle presumably is located closer to the other
end of the pore. Also, the existence of intrinsic voltage dependence of
the unblocking step suggests that the inactivating particle is
effectively charged and is bound to a receptor not located at electric
distance ~0 from the internal pore mouth.
Gomez-Lagunas and Armstrong (1994) proposed that this
recovery-enhancing K+ binding site may be located deep in
the pore (electric distance > 0.5 from outside) because
Ca2+, which blocks Shaker channels with an electric
distance of 0.5, cannot enhance recovery from inactivation, whereas
Cs+, which blocks Shaker channels with an electric distance
of 0.9 from outside, shows remarkable enhancement effect. However,
Ca2+ might reach the enhancement site but could not bind to
it well, and Cs+ might bind to a superficial site to
enhance recovery before it binds to another, deeper site to block the
pore. A superficial location of the enhancement site therefore is not
incompatible with previous reports and seems to be the simplest way to
explain the findings here.
FOOTNOTES
Received Jan. 3, 1997; revised Feb. 24, 1997; accepted Feb. 27, 1997.
This work was supported by Grant NSC 85-2331-B-002-154 from the
National Science Council, Taiwan, Republic of China. I am very grateful
to Dr. Bruce P. Bean for his kind advice and encouragement as well as
for his generous support in equipment. I also thank Dr. Roderick
MacKinnon for providing the Shaker GH4 cDNA as a gift.
Correspondence should be addressed to Dr. Chung-Chin Kuo, Department of
Physiology, National Taiwan University College of Medicine, No. 1, Jen-Ai Road, First Section, Taipei 100, Taiwan, Republic of China.
REFERENCES
-
Armstrong CM
(1971)
Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons.
J Gen Physiol
58:413-437[Abstract/Free Full Text].
-
Armstrong CM
(1981)
Sodium channels and gating currents.
Physiol Rev
61:644-683[Free Full Text].
-
Armstrong CM,
Bezanilla F
(1977)
Inactivation of the sodium channel. II. Gating current experiments.
J Gen Physiol
70:567-590[Abstract/Free Full Text].
-
Baukrowitz T,
Yellen G
(1995)
Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms.
Neuron
15:951-960[ISI][Medline].
-
Bezanilla F,
Perozo E,
Papazian DM,
Stefani E
(1991)
Molecular basis of gating charge immobilization in Shaker potassium channels.
Science
254:679-683[Abstract/Free Full Text].
-
Demo SD,
Yellen G
(1991)
The inactivation gate of the Shaker potassium channel behaves like an open channel blocker.
Neuron
7:743-753[ISI][Medline].
-
Gomez-Lagunas F,
Armstrong CM
(1994)
The relation between ion permeation and recovery from inactivation of ShakerB K+ channels.
Biophys J
67:1806-1815[Abstract/Free Full Text].
-
Heginbotham L,
MacKinnon R
(1993)
Conduction properties of the cloned Shaker K+ channel.
Biophys J
65:2089-2096[Abstract/Free Full Text].
-
Hille B
(1992)
Potassium channels and chloride channels.
In: Ionic channels of excitable membranes, pp 116-121. Sunderland, MA: Sinauer.
-
Hoshi T,
Zagotta WN,
Aldrich RW
(1990)
Biophysical and molecular mechanisms of Shaker potassium channel inactivation.
Science
250:533-538[Abstract/Free Full Text].
-
Hoshi T,
Zagotta WN,
Aldrich RW
(1991)
Two types of inactivation in Shaker potassium channels: effects of alterations in the carboxy-terminal region.
Neuron
7:547-556[ISI][Medline].
-
Iverson LE,
Rudy B
(1990)
The role of divergent amino and carboxyl domains on the inactivation properties of potassium channels derived from the shaker gene of Drosophila.
J Neurosci
10:2903-2916[Abstract].
-
Kamb A,
Iverson LE,
Tanouye MA
(1987)
Molecular characterization of shaker, a Drosophila gene that encodes a potassium channel.
Cell
50:405-413[ISI][Medline].
-
Kuo C-C,
Bean BP
(1994)
Sodium channels must deactivate to recover from inactivation.
Neuron
12:819-829[ISI][Medline].
-
Kuo C-C,
Hess P
(1993)
Characterization of the high-affinity Ca2+ binding sites in the L-type Ca2+ channel pore in rat phaeochromocytoma cells.
J Physiol (Lond)
466:657-682[Abstract/Free Full Text].
-
MacKinnon R,
Yellen G
(1990)
Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels.
Science
250:276-279[Abstract/Free Full Text].
-
Murrell-Lagnado RD,
Aldrich RW
(1993)
Energetics of Shaker K channels blocked by inactivation peptides.
J Gen Physiol
102:977-1003[Abstract/Free Full Text].
-
Perozo E,
MacKinnon R,
Bezanilla F,
Stefani E
(1993)
Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels.
Neuron
11:353-358[ISI][Medline].
-
Rudy B
(1988)
Diversity and ubiquity of K channels.
Neuroscience
25:729-749[ISI][Medline].
-
Ruppersberg JP,
Frank R,
Pongs O,
Stocker M
(1991)
Cloned neuronal Ik(A) channels reopen during recovery from inactivation.
Nature
353:657-660[Medline].
-
Woodhull AM
(1973)
Ionic blockage of Na+ channels in nerve.
J Gen Physiol
61:687-708[Abstract/Free Full Text].
-
Zagotta WN,
Hoshi T,
Aldrich RW
(1990)
Restoration of inactivation in mutant Shaker potassium channels by a peptide derived from ShB.
Science
250:568-571[Abstract/Free Full Text].
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![<UP>OB</UP>(t)=<UP>OB</UP>(0) · <UP>exp</UP>[−(&agr;+&ggr;) · t],](/content/vol17/issue10/fulltext/3436/img003.gif)
![znid><UP>O</UP>(t)={&agr; · <UP>OB</UP>(<UP>0</UP>)<UP>/</UP>[<UP>&dgr;</UP>−(&agr;+&ggr;)]} · {<UP>exp</UP>[−(&agr;+&ggr;) · t]](/content/vol17/issue10/fulltext/3436/img004.gif)
![<UP>C</UP>(t)+<UP>O</UP>(t)=[&agr; · <UP>OB</UP>(<UP>0</UP>)<UP>/</UP>(&agr;+&ggr;)] · {1−<UP>exp</UP>[<UP>−</UP>(<UP>&agr; + &ggr;</UP>) · t]}.](/content/vol17/issue10/fulltext/3436/img006.gif)
