From the * Department of Anesthesiology, Department of Molecular and Medical Pharmacology, and § Department of Physiology,
University of California, Los Angeles, Los Angeles, California 90095-1778; and
Conicet, Buenos Aires, Argentina 1033
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ABSTRACT |
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Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F
channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with
changes in the kinetics of gating current known as "charge immobilization" (Armstrong, C.M., and F. Bezanilla.
1977. J. Gen. Physiol. 70:567-590.). Shaker H4 W434F gating currents are very similar to those of the conducting
clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge
return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at 90 mV return potential changed from a single
fast component to at least two components, the slower requiring ~200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current
(Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both
recoveries are similar at
120 and
90 mV. In contrast, at higher potentials (
70 and
50 mV), the gating
charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of "parallel" inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus
region.
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INTRODUCTION |
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Voltage-activated potassium channels play a key role in
the control of membrane excitability. As potassium has
a negative reversal potential, the opening of these
channels draws the membrane potential toward negative values and far from the excitability threshold. Different activation and inactivation kinetics allow them to
control the repolarization after an action potential (delayed rectifiers, slow inactivation) or both the potential
duration and firing frequency (A-type channels, fast
and slow inactivation). In fast-inactivating Shaker K+
channels, the gating charge return after a depolarizing
pulse is very slow (Bezanilla et al., 1991). This slow return of charge, referred to as "charge immobilization,"
was first seen in the squid axon Na+ channels and correlated with the fast inactivation process by Armstrong
and Bezanilla (1977)
. They proposed a "ball and chain" model, where an inactivation particle binds to the inner mouth of the pore and slows down the gating
charge return. Through the suppression of fast inactivation by the amino terminus deletion mutant and its
restoration by application of the synthetic NH2 terminus (ball peptide) on the cytoplasmic side of the channels, the ball and chain theory was established for
Shaker K+ channels. The cytoplasmic NH2 terminus of
each of the four subunits is responsible for the fast inactivation process (Hoshi et al., 1990
; Zagotta et al.,
1990
; MacKinnon, 1991
; MacKinnon et al., 1993
).
Shaker K+ channels are good candidates to study fast
inactivation and gating current kinetics due to their
high level of expression (Tempel et al., 1987; Iverson et
al., 1988
; Kamb et al., 1988
; Bezanilla et al., 1991
; Stefani et al., 1994a
). The amino deletion mutation
6-46
(Hoshi et al., 1990
) removes fast inactivation without
visible effects on activation, so the channel behavior can be compared in the presence and absence of the
fast inactivation process. Moreover, the introduction of
the W434F pore mutation in the
6-46 Shaker K+ channel results in a nonconducting channel with gating currents very similar to those of the conducting clone. It
allows the study of gating currents without significant
contamination by ionic current (Perozo et al., 1993
;
Starkus et al., 1997
; Yang et al., 1997
). In this study, we
expanded the initial results of Bezanilla et al. (1991)
that
showed charge immobilization in Shaker H4 (ShH4) channels. We correlated the installation and recovery
of the fast inactivation process with changes in the kinetics of gating currents. The experiments were performed in the conducting and nonconducting (W434F)
clones, since the W434F mutation in the wild-type
Shaker K+ channel leads to a channel with gating currents very similar to the conducting one, as previously
reported for Shaker H4
6-46 (ShH4
6-46) and ShH4
6-46 W434F channels (Perozo et al., 1993
). We were
able to monitor the full gating charge return over a
wide range of potentials (from
120 to 6 mV). To correlate fast inactivation and charge movement kinetics,
we compared the time course of fast inactivation with
those of the components of the OFF gating currents. Demo and Yellen (1991)
proved in single channel experiments that, in high external potassium, most if not
all the recovery of the ionic current takes place through
the inactivated (I)1 to open (O) state transition, and proposed that a similar situation may occur at low external
K+ concentrations. However, ionic and gating current
measurements of Shaker K+ channels could be explained
by the existence of a "parallel" pathway of inactivated
states (Zagotta and Aldrich, 1990
; Bezanilla et al., 1991
).
We report here that a sequential model with a single inactivated state cannot fully explain our data, which are
consistent with the existence of a set of parallel inactivated states.
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MATERIALS AND METHODS |
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Molecular Biology, Oocyte Preparation, and Injection of cRNAs
Four different clones of Shaker K+ channels were used: Shaker H4
(ShH4) (Kamb et al., 1988; Tempel et al., 1987
), the noninactivating
6-46 deletion mutant Shaker H4
6-46 (ShH4
6-46)
(Hoshi et al., 1990
) and the two nonconducting versions with the
W434F pore mutation (ShH4 W434F and ShH4
6-46 W434F).
RNA was synthesized from each cDNA construct by linearization
of the pBluescript plasmids (Stratagene Inc., La Jolla, CA) with
EcoRI and transcription with T7 RNA polymerase (Promega,
Madison, WI) using the mMESSAGE mMACHINE kit (Ambion Inc., Austin, TX).
Xenopus laevis (Nasco, Modesto, CA) oocytes (stage V-VI) were used. 1 d before injection of the cRNA, the oocytes were collected and treated with collagenase (200 U/ml; GIBCO BRL, Grand Island, NY) in a Ca2+-free solution to remove the follicular layer. They were injected with 50 nl of cRNA at 0.05-1 µg/µl using a "nano-injector" (Drummond Scientific Co., Broomall, PA). They were maintained at 19°C in an amphibian saline solution supplemented with 50 mg/ml gentamicin (GIBCO BRL) for 2-7 d before experiments.
Solutions and Electrophysiological Recordings
Electrical measurements were performed with the cut-open oocyte vaseline gap (COVG) technique (Stefani et al., 1994a; Bezanilla and Stefani, 1998
; Stefani and Bezanilla, 1998
). The intracellular electrode was filled with 2.7 M Na-methanesulfonic acid
(MES), 10 mM NaCl, 10 mM EGTA, and 10 mM HEPES. Internal
and external solutions were made by mixing stock isotonic (240 mOsm) solutions of the main cation buffered at pH 7.0 with 10 mM HEPES. A typical external solution was 105 mM NaMES, 2 mM Ca(MES)2, 2 mM KMES, 0.1 mM ouabain, and 10 mM
HEPES; it is abbreviated as NaMES Ca2 K2. NaMES Ca2, KMES
Ca2, or N-methyl-D-glucamine (NMDG)-MES Ca2 were also used.
The electrical access to the cytoplasm of the oocyte was achieved
by applying in the bottom chamber a K-Glu (110 mM K-glutamate,
10 mM HEPES), NMDG-Glu (110 mM NMDG-glutamate, 10 mM
HEPES) or NMDG-MES (106 mM NMDG-MES, 10 mM HEPES,
2 mM MgCl2, 0.1 mM EGTA) solution supplemented with 0.1%
saponin. The bottom chamber was then filled with K-Glu, NMDG-Glu, or NMDG-MES. Two different methods were used to deplete
internal K+ in the oocytes: (a) they could be internally perfused
(0.4-1 ml/h) with NMDG-MES or NMDG-Glu with a glass pipette
(20-50-µM diameter at the tip), inserted into the bottom of the
oocyte and connected to a syringe pump; and (b) they were given
repetitive long depolarizing pulses with frequent washes of the
top and guard chambers with NMDG-MES Ca2. Experiments
were performed at room temperature.
Data were acquired with an IBM compatible personal computer,
using a DMA interface and PCLAMP software (Axon Instruments, Foster City, CA). Analog signals were filtered at one-fifth the sampling frequency. Linear capacity and resistive components were digitally subtracted using a P/4 (subtracting holding potential [SHP] =
120 or
90 mV) or a P/4 (SHP = 20 mV) protocol. The
adequacy of the subtraction protocol was tested by using different
SHP and comparing unsubtracted records and records subtracted
on and off line. Unsubtracted charge movement could be recorded
after analog compensation of the linear capacity and resistive components at either
120 or 20 mV, potentials at which the gating
charge has practically moved entirely in one direction or the other.
Simulation
The model predictions were obtained using the SCOP 3.5 simulation package (Simulation Resources, Inc., Berrien Springs, MI). The source code was compiled with the DJGPP C/C++ compiler
(Delorie Software, Rochester, NH) on an IBM PC compatible
computer. The activation pathway corresponds to the model previously proposed to account for ShH4 6-46 W434F gating currents (Bezanilla et al., 1994
), with slightly different parameters as
shown in Table I. The generated data were filtered at the same
cut-off frequency as the experimental data using a Gaussian filter
(Colquhoun and Sigworth, 1983
). The voltage-dependent rates
are assumed to be of the form
=
0exp(z
eV/kT) for the forward rates
and
=
0exp(
z
eV/kT) for the backward rates
,
where
0 and
0 are the rates at 0 mV, and z
and z
are the equivalent charges moving up to the transition state.
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RESULTS |
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Ionic and Gating Currents in ShH4 and ShH4
6-46 Channels
The general properties of ionic and gating currents of
ShH4 channels are illustrated in Fig. 1. During the depolarizing pulses, ShH4 channels have fast inactivating
ionic currents (Fig. 1 A), while ShH4 6-46 have sustained currents (Fig. 1 B). The fast inactivation in ShH4
is due to the NH2 terminus of the subunits of the homotetramer, as shown by Hoshi et al. (1990)
by deletions in this domain.
|
To record gating currents, ionic currents must be
eliminated. To this end, in the conducting clones, we
internally perfused the oocytes with NMDG-MES or
NMDG-Glu. Fig. 1 shows unsubtracted gating currents
(solid lines) and the corresponding time integral (dashed
lines) for ShH4 and ShH4 6-46 (Fig. 1, C and D, respectively) from
90 mV holding potential (HP) to various
depolarizing potentials.
In the range of potential studied (120 to 50 mV),
ON gating currents are indistinguishable between the
two clones. From the holding potential (
90 mV),
small depolarizations or hyperpolarizations generate
fast rising gating currents with a single exponential decay. For depolarizations more positive than
50 mV,
where the open state becomes populated, the ON develops a pronounced rising phase (not seen at this sampling frequency) and a slow second component appears in the decay phase. With increasing depolarizations, the slow ON component becomes faster and
progressively merges with the initial fast component in
such a way that the two components become indistinguishable at potentials more positive than
10 mV
(Stefani et al., 1994a
). At
90-mV return potential, the
OFF gating currents of the two clones are indistinguishable for potentials more negative than
50 mV in
which the open state is not reached. They have an instantaneous rising phase followed by a single exponential decay. For potentials more negative than
50 mV,
gating currents can be recorded in K+-containing solution without significant contamination from the ionic
current as the channel open probability is very low. They are indistinguishable from those recorded in the absence
of K+ in NMDG-MES-perfused oocytes (data not shown).
A salient difference between both clones appears in
the OFF gating currents for pulse potentials more positive than 50 mV. In both cases, they show a rising
phase followed by a slower decay phase, which is much
slower for ShH4 channels than for ShH4
6-46 channels (compare Fig. 1, C and D). In the noninactivating
clone, the gating charge has recovered in 10-20 ms,
while in the fast-inactivating clone, it has not completely recovered after 100 ms, as noted from the time
integral. This slow charge return referred to as charge
immobilization makes it difficult to study the OFF gating currents, since they can be easily contaminated by
residual tail or leakage currents. For this reason, we
used the pore mutation W434F, which prevents ionic
conduction without apparently affecting the gating currents in ShH4
6-46 channels (Perozo et al., 1993
). Fig.
2 shows gating currents (solid lines) and the corresponding time integral (dashed lines) for both clones
with the W434F pore mutation (ShH4 W434F [A] and
ShH4
6-46 W434F [B]). The gating currents in the
corresponding conducting and nonconducting clones have similar properties. As in the conducting clones,
the main difference between the inactivating and noninactivating clones is that, after depolarizations that
populate the open state, the OFF gating currents show
a fast and slow phase of decay that is more pronounced
in the inactivating clone. We will hereafter refer to the
fast returning charge as Qf and to the slow returning
charge as Qs.
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Recording of the Total Charge Return in ShH4 W434F
Gating currents are capacitive currents; therefore, the
ON and OFF gating charges (QON and QOFF, respectively) must be equal (Figs. 1 and 2). The ON and OFF
equality can be easily verified for the noninactivating
Shaker clones since the gating charge fully returns in
<20 ms at 90 mV, as shown by the time integral. Longer OFF pulse durations are necessary to observe
the full charge return in the inactivating clone due to
the charge immobilization. Previously reported fractions of the total charge that was recovered at
90 mV
were ~0.5 for 30 ms after repolarizations to
90 mV (Bezanilla et al., 1991
; Stefani et al., 1994b
). In this
work, we recorded unsubtracted gating currents on a
longer time scale for which the complete return of the
OFF charge could be monitored (Fig. 2 A). QON and
QOFF measured every 2 mV from
120 to 6 mV are plotted in Fig. 3 versus the membrane potential. For potentials higher than
35 mV, there is a small difference
between QON and QOFF that can be accounted for by a
limited integration interval.
|
Components of Gating Charge Returning from Closed, Open, and Inactivated States
At potentials around 30 mV, the difference between
the two nonconducting clones ShH4 W434F and ShH4
6-46 W434F is evident when superimposing both OFF
gating currents at
90 mV return potential (Fig. 4).
The initial time course of decay is identical in both clones (Fig. 4 1). This initial phase is probably due to
the gating charge returning from channels that were
still in a closed state. In fact, for more negative pulse
potentials in which the channels remained closed, OFF
gating currents in both clones have a similar time
course to this initial phase. Fig. 4 shows that, after this initial component (1), there is a slow component that is
much slower in ShH4 W434F (3) than in ShH4
6-46
W434F (2). The slow component in ShH4 W434F (Fig.
4 3) should correspond to charge returning from both
open and inactivated channels, while in ShH4
6-46
W434F (2) it should correspond to charge returning
mainly from open channels.
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Separation of Two Components in the ShH4 W434F OFF Charge
To quantify Qf and Qs, which corresponds to the fraction of "immobilized" charge, we fitted the decay phase
of OFF gating currents at 90-mV return potential with
two time constants. The fit was adequate, maintaining
the same time constant for the slow component for different pulse depolarizations ranging from
50 to 2 mV
(Fig. 5 A), indicating that the rate of the slow component does not depend on the preceding depolarizing
potential. For pulse potentials more negative than
50
mV, we considered that Qf was the only component of
the gating charge.
|
The separation of the two components Qs and Qf is illustrated in Fig. 5 A for the OFF gating currents at 90
mV after a pulse to
30 mV. The two components Qf
and Qs and the total QOFF as functions of the membrane potential are plotted on Fig. 5 B. We simultaneously fitted Qf and Qs to a single Boltzmann distribution and QOFF to the sum of these distributions. The fitted values were Vf =
59 mV, zf = 3.2; Vs =
40.7 mV,
zs = 6, where Vf and Vs are the half activation potentials
and zf and zs are the effective valences for the fast and
slow components, respectively. Qf represents 15-20%
of the total charge. This proportion of Qf, which may
correspond to charge movement return from open and
closed channels, is too large for a strictly sequential
model in which the last inactivated state becomes maximally populated.
The Proportion of the Fast Returning Charge Depends on the Repolarization Potential
To test a sequential model with only one inactivated state
(··· C
O
I), we investigated whether the ratio between Qf and Qs depends on the return potential from a
constant depolarizing pulse. The sequential model predicts that the speed of the return of the charge, but not
the proportion between Qf and Qs, should depend on
the repolarization potential. Fig. 6 A shows the voltage dependence on the return potential after a 100-ms test
pulse to 0 mV for the total QOFF, Qf and Qs. The graph in
Fig. 6 B shows that the fraction of Qf is larger at more
negative repolarization potentials, thus making a linear
sequential model for inactivation unlikely.
|
Voltage Dependence of Charge Return at Different Times
It has been shown in Figs. 1 and 2 that large depolarizing pulses (e.g., 0 mV) that populate the inactivated
state greatly slow down charge return. On the other
hand, smaller depolarizing pulses that do not fully inactivate the channels, and populate open and closed
states, have a much faster return of the charge. As illustrated in Fig. 7 A, the consequence is that a larger
amount of charge returns in the initial milliseconds of
repolarization after a small depolarization (pulse to
40 mV) compared with large depolarizing pulse to 0 mV, though the total charge moved is smaller at
40
mV than at 0 mV. Note that the time integrals crossed
~8 ms after repolarization. This behavior is illustrated
in Fig. 7 B, in which we measured QOFF at different
times of integration after the repolarization. For integration times shorter than 8 ms, the charge-voltage
curve peaks at ~
45 mV, and then decays for larger
depolarizing potentials, as expected from slowdown of
the OFF gating as the inactivated state becomes populated.
|
Charge Immobilization and Ionic Currents Inactivation Have the Same Time Course
Charge immobilization has been interpreted as a consequence of the binding of the inactivation particle to
its docking site in the internal mouth of the channel,
preventing the channel from undergoing the deactivation transitions (Bezanilla et al., 1991). The evolution
of the OFF gating currents after a depolarization of increasing duration supports this hypothesis. This is illustrated in Fig. 8 A: we fitted the decay phase of the OFF
gating currents after pulses to 0 mV of different duration with a two-exponential function. Fig. 8 B shows the
plot of the Qf/QON ratio as a function of the ON pulse
duration. The plot shows a single exponential decay
with a time constant of 3-5 ms, which correlates with
the inactivation time constant of the ionic current of
ShH4 at 0 mV (Fig. 9 shows ionic currents for a pulse to
20 mV with a similar time constant). This result supports the link between ionic current inactivation and
the slowdown of charge movement.
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|
Internal Potassium Does Not Affect Inactivation Kinetics
External K+ is known to lower the apparent affinity of
the inactivation particle for its binding site by a "knock-off" mechanism (Demo and Yellen, 1991). Internal K+
may also have some influence on fast inactivation; for
instance, as the W434F mutation disrupts conduction,
it may influence the occupancy of an internal K+ binding site(s) and thus modify the kinetics of ball binding. To assess this possibility, we monitored the ionic currents of conducting ShH4 channels while internally
perfusing the oocytes with NMDG-Glu, giving a pulse to
20 mV every 5 s. The inactivation time constant was
practically unaffected by lowering internal K+ (Fig. 9).
Recovery from Inactivation of Ionic and Gating Currents in Low External K+
The comparison of the recovery time courses of the
ionic and gating currents gives us more insight on the
inactivation mechanism and the underlying kinetic
scheme, through the voltage dependency of the transition(s) involved and the number of components. ShH4 W434F channels have identical gating currents in low
(0 or 2 mM) and isotonic external potassium. In a consistent way, their recovery from inactivation is not affected, though the recovery of the ionic currents is
faster in isotonic potassium than in low external potassium (Demo and Yellen, 1991). This supports the view
that external K+ destabilizes the binding of the "ball
peptide" to the inner mouth of the channel. This effect
was not observed in ShH4 W434F mutant, possibly due
to lack of conduction that prevents the accessibility of
external K+ to the inner mouth. Thus, the time course
of recovery of charge movement in ShH4 W434F may
correspond to a recovery in low external K+ for the
conducting clone, and the recovery experiments were
performed accordingly in 2 mM external K+.
Fig. 10 A shows the protocol to obtain the time
course of recovery of the ionic (ShH4) and gating
(ShH4 W434F) current after a 50-ms prepulse to 20 mV. Fig. 11 shows the different time course of recovery
of the ionic and gating currents for different recovery potentials. For negative recovery potentials (120 and
90 mV), there is a good correlation in the recovery time
course of ionic and gating currents. On the other hand,
at more positive recovery potentials, the gating currents
recover at a much slower rate than ionic currents.
|
|
The ionic current recovery has a two-exponential time
course with a predominant fast component (Demo and
Yellen, 1991). The fast component is exponentially dependent on voltage (effective valence z
= 0.39, n = 13-15, Fig. 12 A,
).
|
Two exponential components are needed to account
for the recovery of the charge. The faster one corresponds to values from 20% (at 50 mV) to 50% (at
120 mV) of the total charge. The voltage dependence
of this component corresponds to a z
of 0.49, n = 4 (Fig. 12 A,
), which is slightly higher than the one
measured from the ionic currents recovery.
The voltage dependence of the slow rate could not
be fitted to a single exponential, indicating that it may
correspond to several rates that make up a composite
pathway of recovery (Fig. 12 B). The data points could
be described by two exponential rates with z values of
1.7 and 0.4. z
= 1.7 corresponds to the z
of the gating
charge recovery in the noninactivating mutant ShH4
6-46 W434F channel, in equivalent experiments (data
not shown).
Recovery of Ionic Currents in Isotonic K+
Demo and Yellen (1991) reported that high external
K+ speeds the recovery from inactivation of the ionic
current of ShH4 channels, which follows a mostly single-exponential time course close to the fast component of the recovery in low potassium. They showed that this recovery occurs mainly through the open state
by studying the single-channel tail currents. In our experiments, the recovery was well described by a single
exponential function, though a two-exponential function gave a better result. Both the rate from the single
exponential fit and the rate of the fast component from
the two-exponential fit are faster than the rate of the
fast component of recovery in 2 mM external K+. The
voltage dependency of both rates was similar but somewhat smaller than the one measured in low potassium
(z
= 0.3 vs. 0.39).
![]() |
DISCUSSION |
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The pore mutation W434F is a powerful tool to study
the gating currents of Shaker K+ channels in K+-containing solutions. Perozo et al. (1993) reported that
ShH4
6-46 W434F channels have gating currents similar to those of ShH4
6-46 channels as recorded in potassium-free solutions using NMDG as the main cation
(Bezanilla et al., 1991
; Perozo et al., 1992
), and that
these mutant channels still undergo a close-to-open transition as shown by the charge immobilization induced by internal TEA (tetraethyl ammonium). We report here that similar results are true for the fast-inactivating ShH4 channel. The charge immobilization induced
by the NH2-terminal inactivation ball is seemingly unaffected by the mutation, and its time course matches the
time course of the ionic current inactivation. The absence of contamination by ionic current allowed us to
monitor the full charge return using long OFF pulses
of 165 ms. This increased resolution helped us to assess
how often the recovery from inactivation must take
place through the open state.
Modeling the Inactivated States
Model 1.
Many aspects of the data are inconsistent with
the predictions of a model with only one inactivated
state in sequence with the open state: ··· C O
IN.
For example, although Model 1A (Fig. 13) predicts
ionic current similar to the data (compare Fig. 14, A
and D), it does not predict the two components of the
ionic current recovery after inactivation (Fig. 15 A). We
tested two versions of Model 1 differing in their activation pathway. The first was a sequential linear model
(Fig. 13, Model 1A, and Table I) derived from Bezanilla
et al. (1994)
. The second model (Fig. 13, Model 1B)
considers four subunits, each with three states (Zagotta et al., 1994
). Both types of models predict very similar
ionic currents. However, they failed to predict the components of the OFF charge movement.
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|
|
|
Model 2.
Baukrowitz and Yellen (1995) demonstrated
that closed-state (C) inactivation is not negligible in
ShH4 even during short depolarizations: pore occlusion by the inactivation ball favors the unbinding of potassium ions from an external binding site whose occupancy influences the stability of the C-inactivated state.
Therefore, a linear model must be extended to consider at least one other inactivated state in series with
the first one: ··· C
O
IN
IC (Fig. 13, Model 2). As
with the two previously described models (1A and 1B),
Model 2 also makes a good prediction of ionic currents
in the
40 to +40 mV range (Fig. 14 B). In addition, it
predicts fairly well the ionic current recovery for the
four studied potentials (
120,
90,
70, and
50
mV), as shown in Fig. 15 B. This model predicts a voltage dependence of the Qf/QOFF ratio closer to the data
than Model 1A (Fig. 15 D); however, it still shows less
voltage dependence than the data. Finally, the OFF gating current kinetics and recovery characteristics shown
in Fig. 16 B are poorly predicted by this model.
Model 3.
It is quite possible that inactivation recovery takes a different route. This can be accounted for
by the addition to Model 2 of a parallel pathway for inactivation recovery, similar to what has been proposed
for the sodium channel inactivation (Armstrong and Bezanilla, 1977; Vandenberg and Bezanilla, 1991
) or for
Shaker fast inactivation (Bezanilla et al., 1991
; Bezanilla
and Stefani, 1996
). The existence of deeper N-inactivated states is supported by the influence of the L382I
mutation on the activation and inactivation kinetics of
Shaker channels, interpreted as enhanced closed-state inactivation (Ayek and Sigworth, 1997
), as well as the
influence of hyperpolarization on the proportions of
fast and slow components of ionic current recovery
(Kuo, 1997
).
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FOOTNOTES |
---|
Address correspondence to Dr. Enrico Stefani, UCLA, Dept. of Anesthesiology, BH-612 CHS, Box 951778, Los Angeles, CA 90095-1778. Fax: 310-825-6649; E-mail: estefani{at}ucla.edu
Received for publication 7 January 1998 and accepted in revised form 5 March 1998.
Dr. Roux's present address is Laboratoire de Neurobiologie, Ecole Normale Supérieure, 75005 Paris, France.We are grateful to Drs. R.S. Hurst and D. Sigg for helpful discussions during the course of this work. We also thank Yuguang Jin for preparing and injecting the oocytes and Zharong Jiang for mRNA synthesis.
This work was supported by National Institutes of Health grants GM-52203 to E. Stefani and L. Toro, GM-30376 to F. Bezanilla, and by an American Heart Association Grant in Aid to R. Olcese. L. Toro is an Established Investigator of the American Heart Association.
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Abbreviations used in this paper |
---|
C, closed state; HP, holding potential; I, inactivated state; MES, methanesulfonic acid; NMDG, N-methyl- D-glucamine; O, open state; SHP, subtracting holding potential.
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REFERENCES |
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1. | Armstrong, C.M., and F. Bezanilla. 1977. Inactivation of the sodium channel. II. Gating current experiments. J. Gen. Physiol. 70: 567-590 [Abstract]. |
2. | Ayek, R.K. Jr., and F.J. Sigworth. 1997. Enhanced closed state inactivation in a mutant Shaker K+ channel. J. Membr. Biol. 157: 215-230 [Medline]. |
3. | Baukrowitz, T., and G. Yellen. 1995. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron. 15: 951-960 [Medline]. |
4. | Bezanilla, F., and E. Stefani. 1996. Gating of the Shaker B potassium channel. In Basic Neuroscience in Invertebrates. H. Koike, Y. Kidokoro, K. Takahashi and T. Kanaseki, editors. Japan Scientific Societies Press. Tokyo, Japan. 3-18. |
5. | Bezanilla, F., and E. Stefani. 1998. Gating currents. Methods Enzymol. In press. |
6. | Bezanilla, F., E. Perozo, D.M. Papazian, and E. Stefani. 1991. Molecular basis of gating charge immobilization in Shaker potassium channels. Science. 254: 679-683 [Medline]. |
7. | Bezanilla, F., E. Perozo, and E. Stefani. 1994. Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation. Biophys. J 66: 1011-1027 [Abstract]. |
8. | Colquhoun, D., and F.J. Sigworth. 1983. Fitting and statistical analysis of single-channel records. In Single Channel Recording. B. Sakmann and E. Neher, editors. Plenum Publishing Corp., New York. 191-263. |
9. | Demo, S.D., and G. Yellen. 1991. The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker. Neuron. 7: 743-753 [Medline]. |
10. | Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1990. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 250: 533-538 [Medline]. |
11. | Iverson, L.E., M.A. Tanouye, H.A. Lester, N. Davidson, and B. Rudy. 1988. A-type potassium channels expressed from Shaker locus cDNA. Proc. Natl. Acad. Sci. USA. 85: 5723-5727 [Abstract]. |
12. | Kamb, A., J. Tweng-Drank, and M.A. Tanouye. 1988. Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron. 1: 421-430 [Medline]. |
13. |
Kuo, C.C..
1997.
Deactivation retards recovery from inactivation in
Shaker K+ channels.
J. Neurosci.
17:
3436-3444
|
14. | MacKinnon, R.. 1991. Determination of the subunit stoichiometry of a voltage-activated potassium channel. Nature. 350: 232-235 [Medline]. |
15. | MacKinnon, R., R.W. Aldrich, and A.W. Lee. 1993. Functional stoichiometry of Shaker potassium channel inactivation. Science. 262: 757-759 [Medline]. |
16. |
Olcese, R.,
R. Latorre,
L. Toro,
F. Bezanilla, and
E. Stefani.
1997.
Correlation between charge movement and ionic current during slow
inactivation in Shaker K+ channels.
J. Gen. Physiol.
110:
579-589
|
17. | Perozo, E., R. MacKinnon, F. Bezanilla, and E. Stefani. 1993. Gating currents from a non-conducting mutant reveal open-closed conformations in Shaker K+ channels. Neuron. 11: 353-358 [Medline]. |
18. | Perozo, E., D.M. Papazian, E. Stefani, and F. Bezanilla. 1992. Gating currents in Shaker K+ channels. Implications for activation and inactivation models. Biophys. J. 62: 160-171 [Abstract]. |
19. |
Roux, M.J.,
D. Sigg,
R.S. Hurst,
F. Bezanilla, and
E. Stefani.
1995.
Modeling of Shaker H4![]() |
20. | Sigg, D., E. Stefani, and F. Bezanilla. 1994. Gating current noise produced by elementary transitions in Shaker potassium channels. Science. 264: 578-582 [Medline]. |
21. | Starkus, J.G., M.D. Rayner, and S.H. Heinemann. 1997. Anomalous conduction in the "non-conducting" Shaker K+ channel mutant W434F. Biophys. J. 72: 232a. (Abstr.) . |
22. | Stefani, E., and F. Bezanilla. 1998. The cut-open oocyte voltage clamp technique. Methods Enzymol. In press. |
23. | Stefani, E., L. Toro, E. Perozo, and F. Bezanilla. 1994a. Gating of Shaker K+ channels: I. Ionic and gating currents. Biophys. J. 66: 996-1010 [Abstract]. |
24. | Stefani, E., L. Toro, E. Perozo, and F. Bezanilla. 1994b. Gating currents of cloned Shaker K+ channels. In Handbook of Membrane Channels. Academic Press, Inc., Orlando, FL. 29-40. |
25. | Tempel, B.L., D.M. Papazian, T.L. Schwarz, Y.L. Jan, and L.Y. Jan. 1987. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science. 237: 770-775 [Medline]. |
26. | Vandenberg, C.A., and F. Bezanilla. 1991. A sodium channel gating model based on single channel, macroscopic ionic, and gating currents in the squid giant axon. Biophys. J. 60:1511-1533. |
27. |
Yang, Y.,
Y. Yan, and
F.J. Sigworth.
1997.
How does the W434F mutation block current in Shaker potassium channels?
J. Gen. Physiol
109:
779-789
|
28. | Zagotta, W.N., and R.W. Aldrich. 1990. Voltage-dependent gating of Shaker A-type potassium channels in Drosophila muscle. J. Gen. Physiol. 95: 29-60 [Abstract]. |
29. | Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1990. Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science. 250: 568-571 [Medline]. |
30. | Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. Shaker potassium channel gating. III: Evaluation of kinetics models for activation. J. Gen. Physiol. 103: 321-362 [Abstract]. |