From the Department of Physiology, Botterell Hall, Queen's University, Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
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4-Aminopyridine (4-AP) binds to potassium channels at a site or sites in the inner mouth of the pore
and is thought to prevent channel opening. The return of hKv1.5 off-gating charge upon repolarization is accelerated by 4-AP and it has been suggested that 4-AP blocks slow conformational rearrangements during late closed
states that are necessary for channel opening. On the other hand, quinidine, an open channel blocker, slows the
return or immobilizes off-gating charge only at opening potentials (>25 mV). The aim of this study was to use quini-dine as a probe of open channels to test the kinetic state of 4-AP-blocked channels. In the presence of 0.2-1 mM
4-AP, quinidine slowed charge return and caused partial charge immobilization, corresponding to an increase in
the Kd of ~20-fold. Peak off-gating currents were reduced and decay was slowed ~2- to 2.5-fold at potentials negative to the threshold of channel activation and during depolarizations shorter than normally required for channel
activation. This demonstrated access of quinidine to 4-AP-blocked channels, a lack of competition between the
two drugs, and implied allosteric modulation of the quinidine binding site by 4-AP resident within the channel.
Single channel recordings also showed that quinidine could modulate the 4-AP-induced closure of the channels,
with the result that frequent channel reopenings were observed when both drugs were present. We propose that
4-AP-blocked channels exist in a partially open, nonconducting state that allows access to quinidine, even at more
negative potentials and during shorter depolarizations than those required for channel activation.
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INTRODUCTION |
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4-Aminopyridine (4-AP)1 blocks many different voltage-gated K+ channels in a variety of tissues including neurons (Ulbricht and Wagner, 1976; Yeh et al., 1976
),
lymphocytes (Choquet and Korn, 1992
), skeletal muscle (Gillespie and Hutter, 1975
), and heart muscle
(Kenyon and Gibbons, 1979
; Simurda et al., 1989
; Castle and Slawsky, 1993
). Block by 4-AP is mediated by its
cationic form from the intracellular face of the channel
(Kirsch and Narahashi, 1983
; Choquet and Korn, 1992
;
Kirsch et al., 1993
; Bouchard and Fedida, 1995
). 4-AP
can bind to either open (Wagoner and Oxford, 1990
;
Choquet and Korn, 1992
; Kirsch and Drewe, 1993
) or
closed (Yeh et al., 1976
; Thompson, 1982
; Kehl, 1990
)
channels and can also be trapped within deactivating
channels at hyperpolarized potentials (Choquet and
Korn, 1992
; Kirsch and Drewe, 1993
; McCormack et al.,
1994
; Bouchard and Fedida, 1995
; Rasmusson et al.,
1995
). Trapping of 4-AP may occur by protection of the
drug binding site by the activation gate (Kirsch and
Drewe, 1993
) so that it cannot be washed out with a
drug-free solution when the channel is closed. Binding
of 4-AP is also thought to be mutually exclusive to both fast (Campbell et al., 1993
; Castle and Slawsky, 1993
;
Stephens et al., 1994
) and slow inactivation (Castle et
al., 1994
; Fedida et al., 1996
). It has also been recently
shown to prevent slow gating charge immobilization associated with C-type inactivation (Fedida et al., 1996
).
The study of K+ channel gating currents can give important additional information on the state dependence of drug action on the channels. K+ channel closure is characterized by a slowed return of gating charge after channel opening (Taglialatela and Stefani,
1993; Stefani et al., 1994
) manifested as a reduced peak
and slowing of the decay of the gating current waveform upon repolarization (Perozo et al., 1993
; Stefani
et al., 1994
). To explain the slowing, models of gating
include either additional slow transitions that carry little charge, before the open state that delays charge return after opening (Taglialatela and Stefani, 1993
; Bezanilla et al., 1994
; Zagotta et al., 1994
b) or a more concerted rearrangement of subunits as a final step to
channel opening, reversal of which slows charge return
on repolarization (Bezanilla et al., 1994
; McCormack et
al., 1994
; Stefani et al., 1994
). 4-AP accelerates the time
course of Shaker K+ channel off-gating currents (McCormack et al., 1994
; Bouchard and Fedida, 1995
), and
this has been interpreted as a prevention of the late
slow steps in channel activation gating that lead to
opening (McCormack et al., 1994
; Bouchard and
Fedida, 1995
). This could then reduce or prevent predominantly open state-dependent inactivation (Campbell et al., 1993
; Castle et al., 1994
; Fedida et al., 1996
).
Quinidine is an antiarrhythmic drug that blocks cardiac Na+ channels to alter action potential duration
and slow conduction (Colatsky, 1982; Clarkson and
Hondeghem, 1985
; Slawsky and Castle, 1994
) and
blocks repolarizing cardiac K+ channels to prolong action potential duration (Colatsky, 1982
; Imaizumi and
Giles, 1987
; Slawsky and Castle, 1994
; Wang et al., 1995
). These antiarrhythmic actions correspond to its
class Ia and class III properties. When expressed in heterologous systems, RHK1 (Yatani et al., 1993
), Kv1.2
(Tseng et al., 1996
) in Xenopus oocytes, and hKv1.5
channels expressed in mouse L cells (Snyders et al.,
1992
; Yeola et al., 1996
) or in HEK cells (Fedida, 1997
) are all blocked by quinidine. Quinidine is suggested to
bind only to open channels and either has extremely
low affinity or is unable to bind to closed channels
(Snyders et al., 1992
; Slawsky and Castle, 1994
; Clark et
al., 1995
). Evidence for open channel block has been
acceleration of K+ current inactivation without effects
on activation or on the steady state voltage dependence
of inactivation (Kehl, 1991
; Slawsky and Castle, 1994
;
Clark et al., 1995
), and the slowing of deactivation tail
currents, which suggests that quinidine has to dissociate before channels can close (Yatani et al., 1993
; Yeola
et al., 1996
; Fedida, 1997
). Detailed studies of quinidine-induced block of hKv1.5 show that block is most
likely mediated at an intracellular site within the internal mouth of the channel through a combination of a
charge-based block and hydrophobic interactions (Snyders et al., 1992
; Snyders and Yeola, 1995
; Yeola et al.,
1996
). Gating currents indicate that quinidine rapidly
blocks only open hKv1.5 channels with a marked slowing of off-gating charge or gating charge immobilization, with little evidence for closed channel block even
at high concentrations (Fedida, 1997
).
Here we attempt to use quinidine, a known open
channel blocker, to probe the state dependence of 4-AP
binding in hKv1.5. Our investigation is centered on gating current measurements and the clear difference in
action between 4-AP and quinidine. The data indicate
that the 4-AP-blocked channels undergo slowing of gating charge return by quinidine, and this effect occurs
even at potentials negative to the threshold of channel
opening. Additional data from single channels strongly
suggest that quinidine has access to channels that have
4-AP bound or trapped in them. We propose that 4-AP
holds the channel in a conformation that is nonconducting but allows quinidine access even at potentials
at which it is normally excluded. A preliminary report
of this work has been published previously (Chen and
Fedida, 1996).
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MATERIALS AND METHODS |
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Cells and Solutions
HEK-293 cells were transiently transfected with hKv1.5 cDNA in
pRC/CMV, using LipofectACE reagent (Canadian Life Technologies, Bramalea, Ontario, Canada) in a 1:10 (wt/vol) ratio.
Transfectants were detected using the phOx system (Invitrogen
Corp., San Diego, CA) as described previously (Fedida, 1997). To
record gating currents, patch pipettes contained 140 mM N-methyl-
D-glucamine (NMDG), 1 mM MgCl2, 10 mM HEPES, 10 mM
EGTA, adjusted to pH 7.2 with HCl. The bath solution contained
140 mM NMDG, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES,
10 mM dextrose, adjusted to pH 7.4 with HCl. In cell-attached patches, to record ionic currents, pipettes contained 135 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 2.8 mM sodium acetate, 10 mM
HEPES, adjusted to pH 7.4 with NaOH. The bath solution contained 135 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM dextrose, pH adjusted to 7.4 with KOH. 4-Aminopyridine was dissolved in distilled water at a stock concentration of 500 mM and
pH-adjusted to 7.4 using HCl. Quinidine (sulfate salt) was dissolved in distilled water at a stock concentration of 5 mM. Experiments using 0.5 or 1 mM quinidine used bath solution at a stock
concentration of 10 mM. 4-AP and quinidine had no effect on
the measured pH of extracellular or intracellular solutions. All
chemicals were from Sigma Chemical Co. (St. Louis, MO).
Electrophysiology
Current recording and data analysis were done using an Axopatch 200A amplifier and pClamp 6 software (Axon Instruments,
Foster City, CA). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments, Inc., Sarasota, FL). After sylgarding and fire polishing, pipettes used to
measure current had resistances of 1.0-2.5 M when filled with
control filling solution. Mean series resistance was 6.8 ± 0.5 M
(n = 29) and mean capacitance was 13.4 ± 1.0 pF. Leakage and
capacitative currents were subtracted on-line using a P/6 protocol
(Zagotta et al., 1994
b). The absence of ionic current at negative
membrane potentials in HEK cells allowed faithful leak subtraction of data. The passive membrane characteristics have been
described in detail when we reported a mean capacity transient
decay time constant of 55.0 µs (Chen et al., 1997
). Capacity compensation and leak subtraction were routinely used, but series
resistance compensation was only rarely used. No difference between results with and without Rs compensation was observed. Gating current data were sampled at 100-330 kHz and low pass filtered at 10 kHz. Single channel data were sampled at 5 kHz and
low pass filtered at 1 kHz. All experiments were performed at 22°C
and cells were superfused continually at a flow rate of 1-2 ml/min.
All Qoff measurements were obtained by integrating the off-gating currents until current waveforms decayed to the baseline. Significant differences between groups of data were tested using Student's t test or ANOVA where appropriate, and a value of P < 0.05 was deemed statistically significant.
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RESULTS |
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Does 4-AP Occlude the Effects of Quinidine on Gating Currents?
In K+ channels, it is well established that 4-AP speeds
Qoff movement (McCormack et al., 1994; Bouchard and
Fedida, 1995
) and quinidine slows the return of Qoff
(Fedida, 1997
). It is thought that 4-AP speeds Qoff and
blocks K+ channels by preventing a final conformational rearrangement before channel opening (McCormack et al., 1994
). On the other hand, quinidine is an
open channel blocker that only binds to channels at
potentials positive to the threshold potential of activation (Snyders et al., 1992
; Clark et al., 1995
; Fedida,
1997
). This has the opposite effect to 4-AP in that the
return of gating charge is slowed more as quinidine impedes closing and channels tend to reblock (Fedida, 1997
). The obvious difference in the effects on Qoff of
these two drugs allowed us to use quinidine as a probe
to test whether 4-AP prevented channel opening and
therefore could occlude the action of quinidine. To obtain data shown in Fig. 1, cells were held at
100 mV in
the presence of 1 mM 4-AP and given a 12-ms depolarizing pulse to +40 mV to activate the channels and
move all gating charge. These gating currents are identical to those observed previously in the presence of
4-AP in both Shaker K+ channels (McCormack et al.,
1994
) and in hKv1.5 (Bouchard and Fedida, 1995
). Igon
represents the movement of gating charge as channels
progress towards the open state, whereas, upon repolarization, Igoff represents the return of gating charge
as the channels deactivate. In the presence of 1 mM
4-AP, Igoff reaches a peak rapidly and decays rapidly.
These gating currents are unlike those seen without 4-AP, which have a slow component of gating charge return that results in a reduction in peak Igoff and slowing
of the decay, on repolarization from potentials positive
to the threshold of channel activation (Stühmer et al.,
1991
; Perozo et al., 1992
; Stefani et al., 1994
; Zagotta et
al., 1994
a). Channel opening and the associated slow
transitions are thought to be prevented by 4-AP (McCormack et al., 1994
; Bouchard and Fedida, 1995
),
which accounts for the rapid rise in Igoff to peak and
subsequent rapid decay to baseline.
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After washing on different doses of quinidine while
pulsing at a rate of 0.5 Hz, a steady state was eventually
reached, usually after 1-2 min. In Fig. 1, the steady
state trace in 4-AP plus quinidine is superimposed over
the trace in 4-AP alone in all panels. 4-AP speeding of
gating charge movement caused a rapid rise and subsequent decay of Igoff while conserving the amount of
charge moved (McCormack et al., 1994; Bouchard and
Fedida, 1995
). Fig. 1 A shows little effect after addition
of 10 µM quinidine, with the labeled arrow indicating
the dose of quinidine and the peak Igoff. With the addition of 50 and 100 µM quinidine (Fig. 1, B and C), an
obvious dose-dependent decrease in the peak Igoff was
evident. At higher doses of quinidine up to 1 mM (Fig.
1, D-F), a slight increase in the rate of decay of the Igon
became apparent, and peak Igoff was further reduced,
but the most striking action of quinidine was to further
slow the time course of Igoff decay. At higher doses,
there was also the appearance of a positive current
"notch" at the beginning of the Igoff that reflects the slow charge return induced by quinidine. It appeared
that in 4-AP plus quinidine, Igoff waveforms were slowed
in a manner similar to that of control without any drugs
(Chen et al., 1997
; Fedida, 1997
), but not to the extent
seen in quinidine alone (Fedida, 1997
). For example,
100 µM quinidine alone reduced peak Igoff almost to
zero (Fedida, 1997
). So it appeared that in the presence of 4-AP, when the channels were depolarized to
potentials at which they should be fully open, the effects of quinidine on Igoff were still present, but somewhat attenuated.
This partial protection of channels from the full action of quinidine by 4-AP was not restricted to high
4-AP concentrations. In Fig. 2, the effect of changing
the 4-AP concentration on the effect of 200 µM quinidine is shown. Preexposure of cells to 4-AP at concentrations from 1 mM down to 50 µM (Fig. 2, A-D) had
quite similar effects. As the 4-AP concentration was lowered, there was some minor slowing of the return of
off-gating current in 4-AP alone, but the cells were still
largely protected from the charge-immobilizing actions
of 200 µM quinidine when it was added. The effect of
quinidine was to slow the return of off-gating charge such that time to peak Igoff was slowed and its amplitude was reduced ~50%. Not only could relatively low
concentrations of 4-AP protect the channels from the
full effects of quinidine, but 4-AP could apparently reverse the actions of quinidine added first. This effect is
shown in Fig. 2 E. Here the cell was exposed to 10 µM
quinidine alone, which reduced Igoff markedly, as
shown by Fedida (1997). Subsequent addition of 100 µM
4-AP reversed this reduction in Qoff and produced a
rapid Igoff waveform.
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When compared with control data with only 4-AP
present (Fig. 3 A, ), experiments with 50 µM (
), 200 µM (
), and 1 mM 4-AP (
) plus quinidine showed
that apart from slowing Igoff, doses greater than 10 µM
quinidine caused a small dose-dependent reduction in
the returning gating charge; that is, a degree of Qoff immobilization. The normalized fractional block of Qoff,
f = (1
Qoff/Qon) was fit with the function:
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(1) |
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where max is the fitted maximum level of block (~0.2
in each case), Kd is the concentration at which there is
a half-maximal effect, [D] is the quinidine concentration, and nH is the Hill coefficient. The Kd for quinidine in the presence of 1 mM 4-AP was calculated to be
144 µM and nH was 1.01. At 200 µM 4-AP, Kd for quinidine was 141 µM, and at 50 µM 4-AP, the Kd was 67 µM. In comparison, with quinidine alone, the Kd was 7.2 ± 1.6 µM and the nH was 0.84 ± 0.21 at a depolarization
of +60 mV (Fedida, 1997). The Kd for quinidine in the
presence of 200 µM or 1 mM 4-AP is ~20× greater than
with quinidine alone, while the nH remains relatively
unchanged (~1). The slopes of the dose-response relations in quinidine and 4-AP suggests that a single binding site exists for quinidine and that cooperativity is not
an important factor in the binding of quinidine to the
channel, either in the absence or presence of 4-AP. At
50 µM 4-AP the Kd was much lower, but still it should
be noted that at all concentrations of 4-AP studied the
maximum immobilization of charge was ~20%. The ratio of the maximum rate of charge returned, compared
with the rate of on-gating charge movement (peak
Igoff/Igon) in 1 mM 4-AP alone is shown in Fig. 3 B (
)
to be ~2, but with increasing doses of quinidine (
),
the ratio decreased from 2.1 at 10 µM (n = 1) to 0.72 ± 0.02 at 1 mM quinidine (n = 4). In 50 µM 4-AP alone,
the off-gating charge movement was not so accelerated
as in 1 mM 4-AP, so that the Igoff/Igon ratio was ~1.6
(
). At this concentration of 4-AP, there was a more
marked reduction of the ratio in the presence of quinidine that was apparent at concentrations as low as 10 µM
(
). Fig. 3 C shows the dose dependence of the time
constant of decay of Igoff (
off). In the paired controls
with 50 µM or 1 mM 4-AP (
), the
off was ~0.4 ms and
significantly increased with the addition of quinidine
(
,
) in doses above 10 µM, reaching 1.23 ± 0.08 ms
in 1 mM quinidine. This increase in
off reflects the
dose-dependent slowing in the decay of Ig off to baseline (Figs. 1 and 2).
At potentials at which channels were fully activated,
quinidine exposure in the presence of a range of 4-AP
concentrations resulted in a significant slowing in the
return of gating charge on repolarization associated
with a reduction in peak Igoff/Igon, and 15-20% immobilization of returning charge. These effects of quinidine are significant but, in the presence of 4-AP, channels appear protected from the larger effects of quinidine alone (Fedida, 1997). At 50 µM, near the Kd for
4-AP alone, somewhat more Qoff immobilization in the
presence of low concentrations of quinidine was noted
(Fig. 3 A). Apart from this, little difference in the action of quinidine was seen across a 20-fold concentration range of 4-AP. This finding suggested that quinidine and 4-AP were not competing for the same binding sites on hKv1.5. Rather, the data suggested that at
potentials at which the channels should be fully activated, both 4-AP and quinidine could access binding
sites on the channel, and that the presence of 4-AP allosterically modulated the quinidine site such that its
effectiveness at charge immobilization was reduced.
The Voltage Dependence of Quinidine Action in 1 mM 4-AP Is Changed
Quinidine has been shown to affect hKv1.5 ionic currents between 30 and 0 mV (during channel opening), suggesting an affinity for the open state (Snyders
et al., 1992
). In confirmation of this, quinidine has also
been shown to slow Igoff only at potentials positive to
25 mV (Fedida, 1997
). If 4-AP prevented channel
opening, we would expect quinidine's effects to be fully
prevented
which was not seen (Figs. 1 and 2). At least
two possibilities exist that could explain these observations: (a) significant numbers of channels can transiently escape 4-AP block, open, and are then blocked
by quinidine; or (b) channels with 4-AP bound exist in a conformationally "open" but blocked state that does
not allow ion conduction, but that allows quinidine access to its binding site. We have tested the two hypotheses by examining gating current waveforms at different
potentials, with a particular emphasis at potentials
where the channel should not be open; that is, negative to the activation threshold at ~
25 mV. Fig. 4 A shows
gating current data collected during 9-ms depolarizing
pulses to between
100 and +60 mV. Transient Igon
can be seen during depolarizations positive to
70 mV
that peak rapidly and decay more quickly during larger
depolarizations, similar to those seen in Shaker K+ channels expressed in Xenopus oocytes (Perozo et al., 1992
;
Stefani et al., 1994
). Igoff in the presence of 4-AP were
uniformly fast after pulses across the entire potential
range and decayed rapidly at
100 mV. This seems to
negate the first hypothesis as Igoff data in the presence
of 4-AP, which represent the behavior of a large population of channels, do not show any Igoff slowing after
pulses to positive potentials (which would be evidence
for channels having opened) compared with pulses
negative to the opening threshold. When 100 µM quinidine was added (Fig. 4 B), the same voltage protocol
showed that Igon remained relatively unchanged, but
that peak Igoff decreased and decayed more slowly, as
seen in the single pulse experiments (Figs. 1 and 2). Superimposed in Fig. 4 B (dotted line) is the gating current
during the pulse to +60 mV, with 1 mM 4-AP alone for
comparison (Fig. 4 A).
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The voltage dependence of the rate of decay of Igon
(on) in 4-AP before (
) and after (
) addition of quinidine is shown in Fig. 4 C. The
on could only be measured at potentials
70 mV after the appearance of
Ig. In 4-AP, the
on at
70 mV was 1.77 ± 0.05 ms and
increased to a maximum of 3.14 ± 0.13 ms at
10 mV,
followed by a voltage-dependent decrease to 0.67 ± 0.07 ms at +70 mV as Igon decayed faster. The curve
with quinidine was similar in shape and had few significantly different points. The differences appeared to
correspond with a small leftward shift of ~10-15 mV.
If slowing of the decay of Igoff is an important indicator of the action of quinidine, the voltage dependence
of this effect can tell us the potentials at which the drug
can access 4-AP-blocked channels. The slowing of the
decay time constant of Igoff (off) at five different
voltage prepulses (
60,
40,
20, 0, and +40 mV) is
shown on an expanded time scale, normalized and superimposed in 1 mM 4-AP before and after the addition of 100 µM quinidine (Fig. 5, A-E). The data are
noisy at
60 mV, but at all potentials that occur negative to (
60,
40 mV), at (
20 mV), and more positive (0 and +40 mV) to the threshold for channel activation, the currents with quinidine decayed much more
slowly than in 4-AP alone. The voltage dependence of
off (Fig. 5 F) showed a striking difference in 4-AP before (
) and after (
) quinidine over the entire voltage range. In 4-AP alone, the
off showed a very slight
increase from 0.48 ± 0.03 ms at a prepulse of
70 mV
to 0.53 ± 0.02 ms at +70 mV. However, after addition
of quinidine, the
off was dramatically slowed ~2-2.5-fold with a larger voltage-dependent increase from 0.90 ± 0.05 ms at
70 mV to 1.17 ± 0.03 ms at +70 mV. The
fact that significant slowing occurred throughout the
voltage range suggests that, in the presence of 4-AP,
quinidine has access to the channels even at potentials
where the channels would normally be closed.
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The effects of quinidine with 4-AP on gating charge
were examined by integrating both Igon and Igoff of the
data traces from Fig. 4, A and B. These integrals are
shown in Fig. 6, A and B as Qon movement for depolarizations from 100 mV to between
80 and +60 mV in
20-mV steps, and as Qoff for return of gating charge upon repolarization to
100 mV. In Fig. 6 A, in 1 mM
4-AP, Qon and Qoff both increased more rapidly and
eventually saturated at ~1.4 pC with larger depolarizations. The Qoff integrals shown here are similar in size and
shape to those previously recorded in the presence of
permeant K+ and Cs+ (Chen et al., 1997
). When 100 µM
quinidine was added to the same cell (Fig. 6 B), Qon behaved similarly in its rise and saturation at 1.4 pC.
However, Qoff did not have the same properties in quinidine, compared with 4-AP alone. Instead, the rise time
was slower and the amount of charge saturated at
~1.26 pC. The +60-mV trace from the 4-AP data (Fig.
6 A) is included for comparison (Fig. 6 B, dotted line).
Compared with 4-AP alone (Fig. 6 C,
), mean normalized Qoff data from depolarizations up to +70 mV in
10-mV steps show a leftward shift in the voltage dependence of charge movement in the presence of quinidine plus 4-AP (
). The Qoff in quinidine plus 4-AP was
normalized to Qoff in 4-AP alone (
) and the resulting Qoff-V Boltzmann curve was shifted left ~13 mV, showing earlier saturation to ~90% of the level in 4-AP
alone (Fig. 6 C, solid lines). The valence (z) decreased
slightly from 2.06 ± 0.03 to 1.89 ± 0.04 e0 after addition
of quinidine (n = 4). Fig. 6 D shows the voltage dependence of the Qoff/Qon ratios from
70 to +70 mV.
Since the amounts of charge moved at more negative
potentials were very small, the ratios varied more as evidenced by larger error bars. There was a trend for a reduction in Qoff/Qon over the entire potential range, but
this was only significant positive to
20 mV, near the
threshold of channel activation. Depolarizations positive to
20 mV showed that, at most, ~10% of Qoff is inhibited, and this amount of fractional charge block for
1 mM 4-AP with 100 µM quinidine is in agreement with
data obtained from the single-pulse dose-response
curve in Fig. 3 A. Although the charge block did not
seem to be complete until more positive potentials, the
leftward shift of the Qoff-V curve in Fig. 6 C, along with
measurements of
off (Fig. 4), all suggest that quinidine
has an effect on gating charge movement throughout
the voltage range in the presence of 1 mM 4-AP.
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The Time-dependence of Charge Return Is Slowed in Quinidine
In Shaker K+ channels, Igoff has a slowed decay and decreased peak with longer depolarizing prepulses up to
8 ms (Bezanilla et al., 1994; Zagotta et al., 1994
a). This
time-dependent slowing is prevented by the addition of
4-AP and return of Qoff remains fast for depolarizations
up to ~350 ms (McCormack et al., 1994
; Fedida et al.,
1996
). Igon and Igoff are shown for a depolarization to
+60 from
100 mV for varying lengths of time in the
presence of 1 mM 4-AP (Fig. 7 A). The first pulse was
0.45 ms long, and subsequent pulses were incremented
by 0.60 ms. Superimposition of the data traces showed
consistent Igon and reflected the full recovery of gating
charge between the pulses at a frequency of 0.5 Hz.
Peak Igoff increased at short depolarizations, saturating after ~2 ms and decay of Igoff remained fast for all
pulses. After addition of 100 µM quinidine to the same
cell (Fig. 7 B), Igon was similar, but Igoff showed a large
difference in both the peak and rate of decay. Off-gating currents showed a blunted peak and slowed decay
even for the shortest depolarizations, and after ~2 ms,
a notch appeared at the moment of repolarization,
which was absent in 4-AP alone. Mean data for peak
Igoff normalized to maximum peak Igon showed that before addition of quinidine (Fig. 7 C,
), the ratio increased after the first prepulse of 0.45 ms and saturated
after 2 ms at a value of ~2.0. When quinidine was
present (Fig. 7 C,
), normalized Igoff was always less
than in 4-AP alone, and saturated at ~0.8 after 2 ms.
This suggested that quinidine was able to affect (reduce
the peak) Igoff very rapidly, and this effect was maintained for depolarizations up to 8 ms duration. The decay rate of Igoff was significantly slowed at all depolarization durations tested in the presence of quinidine. In
Fig. 7 D, single exponential fits of Igoff showed that in
4-AP alone (
),
off was ~0.4 ms. In contrast, addition
of quinidine (
) significantly slowed
off ~2.5-fold to
~1.0 ms. Similar to the voltage-dependent effects of
quinidine in the presence of 4-AP, the time-dependent
effects consisted of a relative reduction in the peak Igoff
and slowing of
off by ~2-2.5-fold. These changes develop during the briefest depolarizations and saturated
after a depolarizing prepulse to +60 mV of ~2 ms in
length.
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By contrast, inhibition of charge movement (Qoff) in
the presence of quinidine plus 4-AP takes time to become established. Fig. 8, A and B, show superimposed
Qon and Qoff for the data in Fig. 7, A and B. In 1 mM
4-AP alone, the Qon and Qoff rose rapidly and saturated
after a +60-mV prepulse of ~3 ms in length. After addition of 100 µM quinidine, similar Qon and Qoff are evident, although the rise of Qoff was slower, and saturation also occurred after a prepulse of ~3 ms duration.
Mean data for Qoff normalized to maximum Qon (Fig. 8
C) show that in quinidine plus 4-AP (), Qoff saturated
after a prepulse of 2.35 ms at ~90% of maximum Qon,
compared with 100% saturation in 4-AP alone (
).
Measurements of Qoff/Qon (Fig. 8 D) show that charge
was conserved in 4-AP throughout (
), but there was a
significant time-dependent loss of Qoff in quinidine
plus 4-AP (
) after a prepulse >1.05 ms. The maximum loss of charge reached ~10% at a prepulse of
7.05 ms, which agrees with data obtained from single-pulse dose-response experiments (Fig. 3 A), and with
depolarizations >
20 mV (Fig. 6 D). Quinidine seemed
to exert an immobilizing effect on Qoff very rapidly
(~1-2 ms) in the presence of 4-AP that did not occur in 4-AP alone. However, quinidine had immediate effects (<1 ms) in reducing peak Igoff (Fig. 7 C) and slowing
off (Fig. 7 D).
|
Quinidine Has Access to a Single Channel Blocked by 4-AP
The data so far suggests that 4-AP-blocked channels are
accessible to quinidine at all potentials and times studied, even those at which the channel is normally closed.
From this we hypothesized that, at the single channel
level quinidine might be able to convert the essentially
silent behavior known to occur in 4-AP (McCormack et
al., 1994) to a flickering type of block seen with quinidine (Yatani et al., 1993
; Tseng et al., 1996
). hKv1.5 single channels have a high Po (Fedida et al., 1993
; Philipson et al., 1993
), so it was relatively simple to identify
single channel patches from the appearance of a single
opening level. Control data from such a patch are
shown in Fig. 9 A. Cells were depolarized in high (135 mM) bath [K+] and pipettes contained 5 mM [K+].
Patches were held at
80 mV and given a depolarizing
pulse of +60 mV for 800 ms at 0.5 Hz. Control traces in
Fig. 9 A are shown after subtraction of blank traces to
eliminate capacity transients. Single channel openings
occurred in bursts of varying duration and within bursts,
the channel was mostly open with frequent, brief closings. An ensemble average of 74 sweeps from the same cell is shown in Fig. 9 B with a dotted line indicating the
baseline and the voltage protocol below to indicate the
timing of the pulse. During addition of 1 mM 4-AP to
the bath (Fig. 9 C), the channel displayed decreased
openings, eventually becoming completely silent in the
steady state. When averaged, 75 sweeps show that the
average current in 4-AP could not be distinguished
from the baseline. After addition of 100 µM quinidine
to the same cell, representative traces in the steady state
show that the single channel openings reappeared (Fig.
9 E). The channel open time was greatly reduced within
bursts and closing events were prolonged. Sweeps with
no openings were rare compared with 4-AP alone. An
ensemble average of 80 sweeps is shown in Fig. 9 F. The
average current was ~50% of the control average current (Fig. 9 B). Similar results with 4-AP and quinidine
were obtained in four other single channel patches.
Clearly, the addition of quinidine, in the continued presence of 4-AP, resulted in channel reopening and the
adoption of a "flickering" open channel block behavior.
|
Open- and closed-dwell time histograms in control
and in the presence of 4-AP and quinidine are shown in
Fig. 10. In control, both were fit with a single exponential function and values of 1.42 ± 0.01 and 0.40 ± 0.12 ms were obtained for mean open and closed times, respectively. In 1 mM 4-AP and 100 µM quinidine, mean
open time was reduced to 0.61 ± 0.07 ms (Fig. 10 C).
This may be compared with the effect of 100 µM quinidine alone where the mean open time was reduced to
0.26 ± 0.12 ms. However, the closed-time histogram was
well-fit by a double exponential with two mean closed
times of 0.47 ± 0.11 and 3.82 ± 0.02 ms (Fig. 10 D). The
first closed time likely corresponds to the closed state observed in control, which had a similar mean closed
time. The second closed time was approximately eight
times longer than the control closed time. Similar effects on shortening mean open time and lengthening
closed events have been recently observed with intracellularly applied quinidine on single Kv1.2 channels expressed in Xenopus oocytes (Tseng et al., 1996).
|
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DISCUSSION |
---|
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---|
The Acceleration of Charge Return
Until recently, only 4-AP was known to prevent the off-charge slowing that accompanies K+ channel opening
(McCormack et al., 1994; Bouchard and Fedida, 1995
).
This action of 4-AP was strong evidence that 4-AP
blocked channels by preventing a final transition before the open state. Now, at least three processes are
known to accelerate charge return in K+ channels. In
Shaker K+ channels, external Ba2+ speeds Igoff and accelerates the return of gating charge upon repolarization (Hurst et al., 1996
, 1997
). Hurst et al. (1996
, 1997
)
suggest that Ba2+ destabilized the open conformation
to accelerate the return of gating charge. Recently, we
have shown that permeant monovalent cations also
speed Igoff and return of gating charge (Chen et al.,
1997
). Internal 4-AP increases the decay rate of Igoff,
thereby enhancing return of gating charge (McCormack et al., 1994
; Bouchard and Fedida, 1995
). McCormack et al. (1994)
hypothesized that 4-AP prevented
channels from opening and noted a 10-15% decrease
in Qoff in the presence of the drug. Here, in the presence of 4-AP we have shown that quinidine, a known
open-channel blocker, can still slow the decay of off-gating currents (Figs. 1 and 2), prevent full charge return in 4-AP-blocked channels (Figs. 3 and 6 D), and
cause the reappearance of currents from silent single
channels (Fig. 9).
Since quinidine is known to only block open channels (Snyders et al., 1992; Clark et al., 1995
; Fedida,
1997
), the 4-AP-blocked channels must be able to exist
in a conformationally open, nonconducting state that
allows quinidine access. Quinidine binds to this state with a lower affinity (Kd = 144 µM, Fig. 3 A) compared
with quinidine alone (Kd = 7.2 µM; Fedida, 1997
) with
a small nonsignificant change in the Hill coefficient
(nH = 1.01 with 4-AP present, Fig. 3 A), from 0.84 ± 0.21 (Fedida, 1997
) in quinidine alone. These differences indicate a parallel shift in the quinidine sensitivity in the presence of 4-AP of ~20-fold, and suggest a
change in the affinity of the quinidine binding site.
100 µM quinidine also reduced peak Igoff (Figs. 4 D and
7 C), and shifted the voltage dependence of
on (Fig. 4
C). Most importantly, quinidine decreased
off ~2.5-fold
(Figs. 5 F and 7 D) in the presence of 1 mM 4-AP at all
potentials studied. The slowing was also observed after shorter depolarizations than in quinidine alone (Fedida,
1997
). These two results suggest that 4-AP-blocked channels are vulnerable to quinidine block at more negative
potentials and at earlier times than the limits set by the
threshold potentials (~25 mV) and the activation time
to opening (~1 ms) required for the action of quinidine alone (Fedida, 1997
).
Quinidine slows Igoff decay at all times and potentials
(Figs. 1, 2, 5, and 7), which indicates access to the channel
when it should be closed. Additional evidence for an action of quinidine at potentials negative to the channel
opening threshold were negative shifts in the potential
dependence of charge movement (Qoff-V, Fig. 6 C) and
the decay rate of Igon (on, Fig. 4 C). The direction of these
shifts and their relatively small magnitude (<10 mV) suggest quinidine access to a region of the channel
where it can exert charge screening effects, which are
not seen in the presence of quinidine alone (Fedida,
1997
). To obtain further evidence for quinidine access
to 4-AP-blocked channels, we performed single channel
experiments (Figs. 9 and 10). The effects of 1 mM 4-AP at +60 mV on the single channels was a change from
bursts of channel opening with a high Po to a complete
loss of conduction with a failure to reopen or conduct at
steady state. The addition of 100 µM quinidine, while
keeping the concentration of 4-AP constant, led to characteristic flickering open-channel block with only a few
blank sweeps. There was a reduction in mean open time
to <50% and the addition of a second mean closed
time that was approximately eight times longer than the
closed time also observed in control currents. Recovery
from 4-AP block was not observed before addition of
quinidine, which suggested that quinidine was also able
to change the state of 4-AP-bound channels, and allow
them to open. The addition of the second mean closed
time could reflect the presence of a 4-AP- and quinidine-blocked state from which return to the open state
is possible, but approximately eight times slower than in
the unblocked state.
Immobilization of Charge
There are several situations where the return of gating
charge is slowed or immobilized. The first example of
charge immobilization observed was associated with
open channel block by the inactivation domain in channels exhibiting N-type inactivation (Armstrong and Bezanilla, 1977; Bezanilla et al., 1991
; Demo and Yellen,
1991
). Quaternary ammonium ions such as TEA (tetraethylammonium) also cause open channel block of K+
channels internally and prevent the conformational closing of the channel (Armstrong, 1971
). It was shown that
internally applied TEA slows return of gating charge
(Bezanilla et al., 1991
; Stühmer et al., 1991
). The action
of quinidine on hKv1.5 channels results in gating charge
immobilization (Fedida, 1997
), similar to that seen with
open channel block by internal TEA and the N-type inactivating peptide (Armstrong and Bezanilla, 1977
; Bezanilla et al., 1991
; Stühmer et al., 1991
). In the presence of
4-AP, quinidine had a partial charge immobilizing effect
(at doses >50 µM), which caused a parallel shift in the
dose-response relation compared with quinidine alone
(Fig. 3 A; compare with Fedida, 1997
). The voltage dependence of charge return showed a leftward shift of ~13 mV in the Qoff-V curve when normalized to values in
both 4-AP and quinidine (Fig. 6 C), and significant immobilization of up to 20% of gating charge was observed
positive to
20 mV (Fig. 3 A). The time dependence of
charge return with quinidine plus 4-AP showed that normalized Qoff was reduced by ~10% (Fig. 8 C) and development of significant immobilization took ~1 ms (Fig. 8
D). In 1 mM 4-AP, the gating currents showed a reduction of peak Igoff (Figs. 3 B and 7 C) and slowing of
off
(Figs. 3 C, 5 F, and 7 D) at quinidine doses higher than
10 µM, and at all potentials and prepulse lengths studied.
However, the partial charge-immobilizing effects of quinidine were both voltage (Fig. 6, C and D) and time (Fig. 8,
C and D) dependent and did not reach the full effect in
the presence of 4-AP (Fig. 3 A; compare with Fedida,
1997
). Interestingly, the conditions for quinidine-induced charge immobilization in the presence of 4-AP coincided
with the threshold (~25 mV) and onset (~1 ms) of channel activation. This suggests that there could be coupling
between full channel opening and the ability of quinidine to immobilize charge, perhaps through improved
access or stronger binding to the channel.
Molecular Determinants of 4-AP Trapping, and Binding Sites of 4-AP and Quinidine
Many studies have reported the phenomenon of 4-AP
trapping in K+ channels. 4-AP binds to closed (Yeh et
al., 1976; Thompson, 1982
; Kehl, 1990
) or open (Wagoner and Oxford, 1990
; Choquet and Korn, 1992
)
channels. It remains trapped in the channels at hyperpolarized potentials with relief of block after wash out
occurring upon subsequent depolarization (Choquet
and Korn, 1992
; Kirsch and Drewe, 1993
; McCormack
et al., 1994
). Trapping of blockers in channels was first
proposed for quaternary ammonium compounds (Armstrong, 1971
). By transplanting regions of Kv3.1 into the less 4-AP-sensitive Kv2.1, the cytoplasmic halves of the
S5 and S6 transmembrane segments were determined
to be important for 4-AP binding and were also implicated in forming part of the inner mouth of the pore
(Kirsch et al., 1993
). The actual site of 4-AP binding
was thought to be guarded by the activation gate structure, which would trap the 4-AP in the closed channel
(Kirsch and Drewe, 1993
). The differences in the S5
segment are at positions 434, 435, and 439 (using
hKv1.5 numbering) and in the S6 segment at 507, 510, 512, and 514. The equivalent residues in hKv1.5 show more sequence homology with Kv3.1 than Kv2.1 and, as
expected, the IC50 for hKv1.5 (50 µM; Bouchard and
Fedida, 1995
) is similar to Kv3.1 (100 µM; Kirsch et al.,
1993
), and quite different from Kv2.1 (18 mM; Kirsch
et al., 1993
).
In a mutational study of Kv1.5, two sites were found
to increase or decrease affinity for quinidine when they
were changed (Yeola et al., 1996). These residues at positions 507 and 514 would occur on the same side of
the
-helical S6 segment, separated by two turns. Quinidine open channel block is thought to involve both
electrostatic and hydrophobic components (Snyders et
al., 1992
; Snyders and Yeola, 1995
). As such, substitutions with greater hydrophobicity at position 507 increased affinity for quinidine. It is important to note
that these two sites are also two of the four sites in the S6
segment that are implicated for differential 4-AP sensitivity in Kv2.1 and Kv3.1 (Kirsch et al., 1993
). This strongly suggests that these two drugs have binding sites
close to one another at the inner mouth of the K+ channel pore. Our data supports this idea as the parallel-shifted dose-response curve of fractional charge block
in quinidine plus 4-AP (Fig. 3 A) suggests allosteric
modification of the quinidine site by the presence of
4-AP; the single channel data (Figs. 9 and 10) suggest
quinidine modification of 4-AP binding to the channel.
Recently, it has been shown through the use of Kv2.1
and Kv3.1 chimeras that the cytoplasmic end of the S5
segment (with the same residue differences as for 4-AP
sensitivity) plays an important role in the rate of the
open to first closing transition (Shieh et al., 1997). This
part of S5 could be a part of the structural component
of an activation gate or lid. Another recent study has discovered the ability of a point mutation in Shaker K+
channels at site 470 (508 in hKv1.5) in the S6 transmembrane segment to confer the ability to trap the
organic blockers decyltriethylammonium (C10) and tetraethylammonium (Holmgren et al., 1997
). They determined that the region in S6 near residue 508 is normally covered by the activation lid when the channel is
closed. This site (508) is within the stretch of eight
amino acids that contain the sites implicated for both
4-AP (507, 510, 512, and 514) and quinidine binding
(507 and 514). Therefore, on a molecular level, the
mechanism for 4-AP trapping and binding of both 4-AP
and quinidine may be mediated by the same region of
eight amino acids that represents two full turns of the S6
-helix. 4-AP trapping also likely involves the cytoplasmic
end of S5, which may form part of the activation lid.
Quinidine Access to 4-AP-blocked Channels
We propose a physical model (Fig. 11) where the activation lid normally shuts the channel when it is closed
at potentials negative to the threshold of channel opening (A). When the lid opens, quinidine is allowed access to the channel and can reach its binding sites (Fig.
11 B). The bound quinidine must dissociate before allowing the activation lid to close the channel. This results in delayed channel closing and characteristic
crossover of deactivating tail currents (Snyders et al.,
1992; Fedida, 1997
). 4-AP binds to sites that are likely
covered by the activation lid and exposed when the
channel opens (Fig. 11 C). When the channel closes,
4-AP remains bound to the channel and is trapped
within. The activation lid now cannot shut completely,
because the 4-AP molecule might occupy some of the
sites that the lid normally covers. Incomplete closure of
the activation lid could be an explanation for our observations that quinidine has access to 4-AP-blocked
channels earlier in time and at potentials negative to
the threshold of channel opening (Fig. 11 C). There
was an obvious slowing of the rate of decay of Igoff at
subthreshold potentials, and earlier than expected compared with the onset of quinidine action without
4-AP. The presence of trapped 4-AP in the channels
seems to allow quinidine access and provides the basis
for our model of the 4-AP-blocked channel in a quinidine-sensitive but nonconducting state, analogous to
the "tense" state proposed by McCormack et al. (1994)
.
Since quinidine binding is voltage dependent and is increased at more depolarized potentials (Snyders et al.,
1992
; Clark et al., 1995
; Fedida, 1997
), quinidine affinity would be expected to be reduced at potentials negative to the threshold of activation. In addition, access to
quinidine could be restricted by the partially closed activation lid and could be enhanced at potentials where
the activation lid would open fully (Fig. 11 D). These
two reasons may explain why significant charge immobilization of quinidine in the presence of 4-AP did not
seem to occur at potentials negative to the threshold of
activation and took ~1 ms to develop. Thus, the voltage-dependent increase in affinity of quinidine or the
opening of the activation lid could allow for greater association of quinidine to the 4-AP-blocked channel at
positive potentials. This increased affinity or enhanced access of quinidine could then confer its charge-immobilizing action. Our gating current observations that
quinidine affinity was greatly reduced in 4-AP, coupled
with the single channel observations that reopenings
were virtually nonexistent in 4-AP alone but were facilitated by the addition of quinidine, strongly support the idea that modification of both quinidine and 4-AP
binding occurred when both drugs were present at the
inner mouth of the channel (Fig. 11 D).
|
Conclusion
Using quinidine as a probe of open channels, our results
suggest that 4-AP-blocked channels actually allow access
to quinidine at potentials and times when the channel
should be closed. The simplest explanation of this result
is that 4-AP actually holds the channel in an open, but
nonconducting state. However, at this point, we cannot
tell whether the channel is in a conformationally open
but nonconducting state or if trapping of 4-AP holds
the internal vestibule of the channel partially open
enough to allow quinidine binding, but not enough to
allow ion conduction past narrower, deeper parts of the
K+ channel pore. The latter explanation is more consistent with the fast off-gating currents on repolarization
(McCormack et al., 1994
), supporting the idea of a slow
final opening transition that carries little charge. The
fact that 4-AP trapping and binding of both 4-AP and
quinidine may all be mediated by the same region at the
inner vestibule is also important for understanding the structure of K+ channels and the precise mechanism of
action of ion channel blockers and therapeutic drugs.
![]() |
FOOTNOTES |
---|
Address correspondence to Dr. David Fedida, Department of Physiology, Botterell Hall, Queen's University, Kingston, Ontario, Canada K7L 3N6. Fax: 613-545-6880; E-mail: fedidad{at}post.queensu.ca
Received for publication 14 August 1997 and accepted in revised form 22 January 1998.
This work was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario (Canada) to David Fedida. Fred S.P. Chen was supported by an Ontario Graduate Scholarship.
![]() |
Abbreviation used in this paper |
---|
4-AP, 4-aminopyridine.
![]() |
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