Ca2+-activated K+ channel inhibition
by reactive oxygen species
Marco A.
Soto1,2,3,
Carlos
González1,2,
Eduardo
Lissi3,
Cecilia
Vergara1,2, and
Ramón
Latorre1,2
1 Centro de Estudios Científicos, Valdivia;
2 Facultad de Ciencias, Universidad de Chile, Santiago; and
3 Facultad de Química y Biología, Universidad de
Santiago, Santiago, Chile
 |
ABSTRACT |
We studied the effect of
H2O2 on the gating behavior of
large-conductance Ca2+-sensitive voltage-dependent
K+ (KV,Ca) channels. We recorded
potassium currents from single skeletal muscle channels incorporated
into bilayers or using macropatches of Xenopus laevis
oocytes membranes expressing the human Slowpoke (hSlo)
-subunit. Exposure of the intracellular side of
KV,Ca channels to H2O2 (4-23
mM) leads to a time-dependent decrease of the open probability
(Po) without affecting the unitary conductance. H2O2 did not affect channel activity when added
to the extracellular side. These results provide evidence for an
intracellular site(s) of H2O2 action.
Desferrioxamine (60 µM) and cysteine (1 mM) completely inhibited the
effect of H2O2, indicating that the decrease in Po was mediated by hydroxyl radicals. The
reducing agent dithiothreitol (DTT) could not fully reverse the effect
of H2O2. However, DTT did completely reverse
the decrease in Po induced by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of
KV,Ca channel activity promoted by DTT suggests that
H2O2 treatment must be modifying other amino
acid residues, e.g., as methionine or tryptophan, besides cysteine.
Noise analysis of macroscopic currents in Xenopus oocytes
expressing hSlo channels showed that H2O2 induced a decrease in current mediated by
a decrease both in the number of active channels and
Po.
KV,Ca channels; H2O2
 |
INTRODUCTION |
MOLECULAR OXYGEN
OCCUPIES an essential role in many of the metabolic processes
associated with aerobic existence. Hydrogen peroxide
(H2O2), superoxide radical anion
(O
), singlet oxygen (1O2),
and hydroxyl radical (·OH) are produced by intracellular metabolism
from a variety of cytosolic enzyme systems. NADPH oxidase and nitric
oxide synthase (NOS) family contributes to oxidative stress due to the
generation of free radicals. Free radicals production is
regulated by an enzymatic defensive system (superoxide dismutase, catalase, glutathione peroxidase) and a nonenzymatic system that includes pyruvate, ascorbate, carotenes, and glutathione. The equilibrium between free radical production and antioxidant defenses determines the degree of oxidative stress (13).
The levels of reactive oxygen species (ROS) are enhanced during
inflammation, radiation exposure, endotoxic shock, and
ischemia-reperfusion. The pathologies that have been attributed
to ROS-induced cell dysfunction include skeletal muscle injury
(27, 30) and myocardial damage during ischemia and
reperfusion. In skeletal muscle, exercise increases the rate of ROS
production. This increase is associated with increased levels of lipid
peroxidation and peroxidation products, low catalase concentrations,
and the presence of high levels of myoglobin acting as a catalyst for
the formation of oxidants (8, 27). Kourie
(19) reviews the effects of ROS when interacting with ion
transport systems.
Calcium- and voltage-sensitive channels of large unitary conductance
(KV,Ca) are distributed in different cells and tissues, where they modulate many cellular processes (20, 21).
Because cytosolic Ca2+ activates KV,Ca
channels, they play an important role in coupling chemical to electric
signaling. KV,Ca channels are present abundantly in
virtually all types of smooth muscle cells, where they control the
resting tone (1, 17, 24). KV,Ca channels are
also redox modulated (10, 38, 39). For instance, oxidizing
agents such as H2O2 promote channel inhibition,
and the reducing agent dithiothreitol (DTT) augments channel activity
(10). The effect of H2O2 on the
KV,Ca channel was studied by DiChiara and Reinhart
(10). They reported that hSlo currents
were downmodulated by the oxidizing agent with a right shift of the
probability of opening (Po) vs. voltage curves
and a decrease in the single-channel Po. In the present study, we examined the mode of action of
H2O2 in detail, with the aim of finding a
mechanistic explanation for its deleterious effects on
KV,Ca channels. We found that 1) the targets of
H2O2 action are located in the intracellular
aspect of the KV,Ca channel; and 2) the
H2O2 effect on the KV,Ca channel
activity is mediated by ·OH. The general conclusion is that redox
modulation most probably involves a disulfide/thiol exchange of thiol
groups of some of the numerous cysteines present in the carboxy
terminus of the human Slowpoke (hSlo) protein.
 |
METHODS |
Planar lipid bilayers and single channel recordings.
Lipid bilayers were made from an 8:1 mixture of 1-palmitoyl, 2-oleoyl
phosphatidylethanolamine (POPE) and 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) in decane (13 mg lipid/ml). This lipid solution was applied across a small hole (0.2-0.3 mm in diameter) made in the wall of a Delrin cup separating two chambers of 3.5 ml
cis- and 0.35 ml trans-containing symmetric salt
solutions of 150 mM KCl, 10 mM
3-[N-morpholino]propanesulfonic acid K+ salt , pH 7, [Ca2+]
5 µM. Bilayer formation was
followed by measuring membrane capacitance. Bilayer capacitance was
measured at the end of each experiment to determine possible changes in
bilayer area and/or thickness. Rat skeletal muscle was used to prepare
tubule T membrane vesicles containing KV,Ca channels as
previously described (22). Membrane vesicles were added
very close to the bilayer. Because depolarizing voltages and
cytoplasmic Ca2+ activate KV,Ca channels, the
internal side of the membrane was defined according to the voltage and
Ca2+ dependence of the channel.
H2O2 from a concentrated stock solution was
added to the indicated concentrations. The solutions were stirred for
30 s, and single-channel current records (3-60 min) were
obtained at a constant applied potential of +60 mV unless otherwise stated.
Comparisons between current records obtained in the different
experimental conditions tested were taken in the same single-channel membrane. Only membranes with a stable Po were used.
Data acquisition and analysis.
The current across the bilayer was measured with a low-noise
current-to-voltage converter (6) connected to the solution through agar bridges made with 1 M KCl. Continuous 3- to 60-min single-channel current records were taped on a video recorder. For
analysis, the current was filtered at 400 Hz with an eight-pole Bessel
low-pass active filter and digitized at 500 µs/point. The electrophysiological convention was used, in which the external side of
the channel was defined as zero potential. The experiments were
conducted at room temperature (22 ± 2°C).
Open and closed events were identified using a discriminator located at
50% of the open-channel current. Dwell-time histograms were
logarithmically binned and fitted to a sum of exponential probability
functions with pClamp 6.0 software (Axon Instrument). Closed dwell-time
histograms were fitted to the sum of two exponential functions.
Po was measured as a function of time after
H2O2 addition and
[H2O2]. For single-channel membranes,
Po was obtained as the time spent in the fully
open current level divided by the total time of the record, usually
60 s. Po values were calculated excluding channel closures lasting >200 ms as these events are due to ion channel blockage induced by the contaminant Ba2+ (9,
25).
Oocyte isolation and RNA injection.
Ovarian lobes were surgically removed from adult female X. laevis (Nasco) and placed in 100-mm petri dishes containing OR-2 solution (in mM: 83 NaCl, 2.5 KCl, 1 MgCl2, 5 HEPES; pH
7.6). To dissociate the oocytes, the lobes were incubated for 60 min at
18°C in OR-2 solution containing 1 mg/ml collagenase (GIBCO BRL).
Dissociated oocytes were placed in ND-96 solution (100 mM NaCl, 2 mM
KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, and
50 µg/ml gentamicin, pH 7.4) and were injected with 50 nl of a
solution of human myometrial cRNA of the hSlo channel
-subunit containing 100 ng/µl. Oocytes were kept at 18°C in an
incubator and were used for the experiments 3-4 days after RNA injection.
Electrophysiology.
Macroscopic currents were recorded in cell-attached macropatches and
excised inside-out patches. Patch pipettes resistance were ~1 M
.
Bath and pipettes contained (in mM): 110 KMES, 10 HEPES, 5 HEDTA (the
affinity of HEDTA for iron is about 100-fold less compared with EDTA),
pH 7.0, and the indicated Ca2+ concentrations. The
acquisition and basic analysis of the data were performed with pClamp
6.0 software (Axon Instruments) driving a 12-bit analog interface card
(Labmaster DMA, Scientific Solutions).
Variance analysis.
A series of current traces were recorded after pulsing to a positive
voltage from the holding potential. The average basal variance at the
holding voltage was subtracted from the variance obtained during the
test pulse. The subtracted variance (
2) was plotted vs.
mean current [I(t)] and the data were fitted using (31)
|
(1)
|
where i is the single-channel current amplitude and
N the number of channels; i was obtained from the
initial slope; N was obtained from the nonlinear
curve-fitting analysis done using Microsoft Excel. The maximum open
probability (P
) was obtained according
to the relation: P
= Imax/iN, where
Imax is the maximum mean current measured in the experiment.
Reagents.
POPE and POPC in chloroform were purchased from Avanti Polar Lipids
(Birmingham, AL). The 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB),
DTT, and n-decane were purchased from Sigma (St. Louis, MO).
The perhydrol 30% of hydrogen peroxide, chloroform, ethanol, and
methanol were purchased from Merck Chemical.
 |
RESULTS |
Effect of H2O2 addition on the
KV,Ca single channels.
Fig. 1, A and B,
shows single-channel current records in the absence (control) and
presence of 23 mM H2O2 added to the external and internal side, respectively. This experiment shows that
H2O2 does not affect the
Po when added to the external side;
Po value remains constant even after 30 min of
H2O2 addition. On the other hand, when the
internal side was exposed to the same [H2O2],
Po decreased from 0.621 ± 0.032 to
0.081 ± 0.022 (n = 5) after just 3 min of
addition (Fig. 1, B and C). However, the unitary
conductance remained constant during the time experiments (see Fig.
1B, insets a and b). At this
[H2O2], channel Po
decreases to a very low value in a few seconds. It is surprising that
H2O2, despite its large membrane permeability
coefficient, is ineffective when applied to the external side. This is
due to the fact that, in these experiments, H2O2 (23 mM) was added to an external
compartment having a volume of 0.35 cm3, and the internal
compartment had a volume of 3.3 cm3. Considering a
H2O2 permeability coefficient of
10
4 cms
1 and a bilayer area of 3.14 × 10
4 cm2, the maximum
[H2O2] that can be reached in the internal
compartment is only about 2 mM, and the time to reach this
concentration is >1,000 h. Even if this volume is restricted by the
unstirred layers (~100 µm in thickness), the time needed to reach a
concentration >4 mM (the smallest [H2O2]
tested; see Fig. 2B) would be
several hours.

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Fig. 1.
Hydrogen peroxide (H2O2) induces
a decreased open probability (Po) value only
from the intracellular side. A, B: single-channel
recordings in the absence (control) and in the presence of 23 mM
H2O2 in the extracellular and intracellular
side, respectively. Records a and b (in
B) are insets of square section. Transmembrane potential was
+50 mV. Arrows indicate the closed state. Data from 4 experiments are
summarized as means ± SD in bar graphs in C and
D, respectively.
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Fig. 2.
H2O2 decreases the
Po of the Ca2+-sensitive
voltage-dependent K+ (KV,Ca) channel.
A: single-channel current records in the absence (control)
and in the presence of 8 mM internal H2O2 after
1, 12, 25, and 50 min of addition. Arrows indicate the closed state.
B: time course of the KV,Ca channel
Po decreases after addition of 4 ( ), 8 ( ), and 23 mM
H2O2 ( ). , Data
taken in the absence of H2O2. Transmembrane
potential was +60 mV. Data are presented as means ± SD for
n = 5. Solid lines are fits to the data using Eq. 4. For 4 mM H2O2, n = 103 and = 8.35 ± 0.13 min; for 8 mM
H2O2, n = 103 and = 2.15 ± 0.30 min. The data obtained using 23 mM
H2O2 were fit to a single exponential with
= 0.62 ± 0.09 min.
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Figure 2A shows single-channel current records from a
KV,Ca channel in the absence (control) and presence of 8 mM
H2O2 added to the internal side. In this
condition, the Po decreased from 0.895 ± 0.041 (control, t = 0) to 0.051 ± 0.012 (n = 5) after 30 min of exposure to
H2O2. The decrease in Po
occurred abruptly after a lag time that was
[H2O2] dependent. With 8 mM
H2O2, the lag time was ~10 min but was almost
absent when 23 mM H2O2 was added to the
internal side. At this concentration, the Po
decreased more than 50% in ~2 min. When
[H2O2] was 4 mM, the channel activity decreased after lag periods longer than 30 min (Fig. 2B).
After 30 min in the presence of 23 mM H2O2, the
decrease in Po could not be reversed either by
extensive washing with an oxidant-free solution (Fig.
3, A and D) or by
increasing [Ca2+] in the internal side to 60 µM (Fig.
3, B and E). In the absence of
H2O2 (t = 0, control), the
Po value was 0.879 ± 0.089 (n = 4), and after 30 min in the presence of 23 mM
H2O2, the Po value decreased to 0.065 ± 0.024 (n = 4). In 20 different single-channel membranes, the effect of
H2O2 was irreversible when
Po
0.01. KV,Ca channels that
reach that low a Po cannot recover with
increasing internal [Ca2+]. At a less dramatic decrease
in Po, the H2O2
increasing internal [Ca2+] reversed effect. For example,
after 4 min of the addition of 18 mM H2O2, the
effect was reversed by increasing the [Ca2+] to 200 µM
in the internal side (Fig. 3, C and F). From
these results it is apparent that the oxidizing reactions go through a
series of reversible steps ending in one or more irreversible steps.
According to the multiple-hit model, the initial hits would have the
effect of increasing the energy that separate closed from open states
shifting the Po-voltage curve to the right.
Control Po can be recovered in this case by
increasing the [Ca2+] or voltage. However, when the
channel-oxidizing reaction is complete, the channel enters in an
absorbent quiescent state that cannot be reversed by an increase in
[Ca2+] or voltage.

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Fig. 3.
Reversibility of the H2O2 effect
on the KV,Ca channel. A: after a control period
in the absence of the oxidizing agent (top),
H2O2 was added to the internal side of the
channel to a final concentration of 23 mM (middle). After
30 s in the presence of H2O2,
Po decreased to 0.105; after several minutes in
the presence of H2O2, the internal compartment
was perfused with 10 volumes of a H2O2-free
solution (bottom). B: experiment done as in
A, except that reversibility was tested by perfusing the
internal compartment with a H2O2-free solution
containing 60 µM Ca2+. C: KV,Ca
channel activity can be recovered if, after 18 mM
H2O2 treatment, the internal compartment is
perfused with a solution containing 200 µM of Ca2+. The
[Ca2+] was 5 µM during the control period and after
addition of H2O2. D, E:
average from 4 different experiments under the conditions described in
A and B (means ± SD), respectively.
F: data from 5 experiments under the conditions described in
C (means ± SD).
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Desferrioxamine and cysteine protect
K+ channel activity against ·OH.
H2O2 in the presence of Fe(II) can generate
·OH via the Fenton reaction (28), where
H2O2 is reduced according to the following scheme: Fe2+ + H2O2
·OH + Fe3+ +
OH. This reaction is
a well-known source of ·OH, and there is evidence that the
iron-mediated production of ·OH is an important source of lipid
peroxidation and oxidation of amino acid residues in proteins
(28).
To test whether the effect of H2O2 in the
channel was mediated by ·OH generated from contaminant
Fe2+ or by the H2O2 itself, we
added desferrioxamine or cysteine to the internal chamber.
Desferrioxamine prevents ·OH formation, and cysteine is an efficient
scavenger of ·OH. Desferrioxamine oxidizes Fe2+ to
Fe3+ in the presence of O2 and, if applied
before H2O2 addition to the chamber, protects
against the formation of ·OH via the Fenton reaction. Figure
4A shows that in the presence
of 60 µM desferrioxamine in the cytoplasmic side of the channel, the
addition of 23 mM H2O2 does not promote a
rundown of Po even after an exposure time of 25 min. Figure 4C shows the average of five experiments in a
bar graph. On the other hand, cysteine (a reducing agent) acts as an
electron donor from sulfhydryl groups. Figure 4B shows
single-channel recordings in the presence of 1 mM cysteine in the
internal side and the average of four experiments in a bar graph (Fig.
4D). Similar to what was observed for desferrioxamine, in
the presence of cysteine, 23 mM H2O2 did not
cause a reduction in the Po even after 25 min of
H2O2 exposure.

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Fig. 4.
Desferrioxamine (DFA) and cysteine (Cys) protect against
the deleterious effect of H2O2. A:
after a control period of 3 min, 60 µM DFA was added to the internal
side of the bilayer. DFA protects against the effect of
H2O2, as can be observed in the third
single-channel record. B: Cys (1 mM) added before
H2O2 (23 mM) protects from the decrease in
Po. C, D: average of 4 experiments (means ± SD) under the conditions described in
A and B, respectively.
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Effect of sulfhydryl groups reducing agents.
Confirming previous reports (10, 38, 39), we found
that channel activity in lipid bilayers was increased by exposure of
the intracellular side to the sulfhydryl (SH) reducing agent DTT (2 mM;
Fig. 5A). For this experiment,
we chose channels with a low Po at the calcium
concentration used (~5 µM). Figure 5, A and
C, shows that Po increased 2.3-fold
after we added 2 mM DTT (0.260 ± 0.096 to 0.608 ± 0.134, n = 4). After DTT addition, the increase in
Po took less than 30 s to reach a steady
state and remained constant during usual recording times (15-30
min). Perfusion of the internal side with a DTT-free solution did not reverse the DTT effect. The effect of 23 mM
H2O2 on channel activity was partially reversed
by adding 2 mM DTT after perfusing the intracellular side with about
ten times the volume of the internal compartment with a
H2O2-free buffer (Fig. 5, B and
D). Po values were: control
Po, 0.896 ± 0.124;
Po in the presence of 23 mM
H2O2, 0.054 ± 0.006; and
Po in the presence of 2 mM DTT, 0.511 ± 0.156.

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Fig. 5.
Dithiothreitol (DTT) can partially recover the activity
of H2O2-modified KV,Ca channels.
A: channels having a Po of ~0.3 at
5 µM Ca2+ were chosen; 2 mM DTT added to the internal
side of the bilayer increased their Po.
B: the addition of 2 mM DTT partially recovers the activity
of a H2O2-modified channel. C: the
average increase in Po was from 0.260 to 0.608, n = 4. D: average recovery for 4 experiments
was 55% of control values.
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Modification of SH groups by DTNB.
To determine if SH residues were involved in channel modulation by
redox agents, we used DTNB. DTNB is a hydrophilic oxidative reagent
that attacks specifically SH groups in proteins in a reaction that
involves a thiol-disulfide exchange mechanism. Figure
6A shows the effect of 2 mM
internal DTNB on channel activity. Po decreased
17-fold (Po = 0.052 ± 0.007) compared
with control (Po = 0.889 ± 0.103).
Channel activity was not restored by withdrawal of DTNB from the
internal side of the channel, suggesting a covalent modification.
However, channel activity was almost fully restored by application of 2 mM DTT to the internal side (Fig. 6, A and B). On
the average, in the presence of 2 mM internal DTT,
Po increased to 0.76 ± 0.13 of the initial
control value. This observation strongly suggests that the observed
inhibitory effect of DTNB is specifically related to the oxidation of
SH groups.

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Fig. 6.
DTT recovers the activity of
5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB)-modified channels.
A: currents recorded in control conditions (top
trace), after 2 mM DTNB was added to the internal side of the channel
(middle trace), and after removing DTNB by washing and
adding 2 mM DTT (bottom trace). B: average of 4 different experiments. Control Po values
decreased from 0.889 to 0.052 with DTNB. Addition of 2 mM DTT to the
cytoplasmic side of the channel increased Po to
0.758. [Ca2+] was 5 µM.
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Effect of H2O2 on the macroscopic current
induced by hSlo channels.
Figure 7 shows the effect of
H2O2 on the macroscopic Kv,Ca
currents expressed in X. laevis oocytes. The addition of
H2O2 to the external side (in the pipette)
caused a decrease of ~5% of the current after 3-5 min of seal
formation. No further changes were observed thereafter (Fig.
7A). Incubation of an inside-out patch for 30 min in the
presence of 8 mM H2O2 reduced the macroscopic current by 60% (Fig. 7B). After 1 h, the current
decreased further to about 20% of the control value. Under these
conditions, we obtained data by directly plotting the peak tail current
amplitude at a constant postpulse potential (
60 mV) and as a function
of the test prepulse potential in symmetrical 110 mM K+
(Fig. 7C). Note that H2O2 addition
at the internal side induces a gradual shift of the tail conductance
vs. voltage curves, with a clear decrease in the maximum conductance.

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Fig. 7.
H2O2 inhibits KV,Ca
channel steady-state currents when added to the intracellular side.
A: macroscopic currents in the cell-attached configuration
were recorded in the range 150 to 140 mV.
H2O2 (8 mM) was added in the pipette.
B: KV,Ca current recorded in an inside-out patch
was recorded in the range 50 to 140 mV at 10-mV increments.
H2O2 (8 mM) was added in the bath. Internal
[Ca2+] = 56 nM. C: conductance
(Gtail) vs. voltage curves for the traces shown
in B. Data were obtained measuring the peak amplitude of the
tail currents after repolarization to 60 mV in the range 40 mV to
250-300 mV. Each point is the average of 5 patches in the
inside-out configuration. The experimental data were fitted to a
Boltzmann equation of the form Gtail = Gmax/{1 + exp[(V1/2 - V)/k]}
where V is the applied voltage,
V1/2 is the half-activation voltage, and
k the slope of the Gtail vs. voltage
curve. Gmax was 3,500, 3,100, 2,750, 2,600, and
2,150 pS for the Gtail vs. voltage curves taken
at 0, 5, 10, 20, and 30 min, respectively.
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DiChiara and Reinhart (10) reported a spontaneous decline
in the macroscopic K+ current induced by hSlo
channels after patch excision that was reversed by 1 mM DTT. We also
found a current rundown after patch excision, but it was much less
pronounced than the one reported by DiChiara and Reinhart. HSlo
macroscopic conductance decreased by about 8% 5 min after patch
excision and then remained constant for periods as long as 1 h
(data not shown). The reason for the difference between our results and
those DiChiara and Reinhart (10) is unclear. To avoid an
overestimation of the channel inhibition induced by
H2O2 due to rundown, we added
H2O2 to the internal solution 5-10 min
after patch excision. Itail(V) is
given by the relationship
|
(2)
|
or
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(3)
|
where i is current amplitude of the single channel,
g is unitary conductance, N the number of
channels, and Po(V) the
voltage-dependent open probability. Single-channel current measurements
showed that g is constant; therefore, the fact that
Gtail decreases may indicate a reduction in the
number of channels and/or a decrease in Po (see
Eq. 3). To obtain a proper determination of the
P
behavior during
H2O2 exposure, we used the nonstationary
fluctuation analysis (14, 31-33). Figure
8, A, D, and
G, shows the time course of average current for a +120-mV
pulse at 0, 10, and 30 min of H2O2 exposure.
The noise fluctuations in Fig. 8, B, E, and
H, have a biphasic time course that is a reflection of the
channel Po during the activation of the ionic
current. The variance vs. current plots in Fig. 8, C and
F, were fitted to Eq. 1. The fitted parameters were i = 37.0 ± 2.3 pA and
N = 156 ± 15 channels in control conditions.
P
, obtained using the relation
P
= Imax/iN, was 0.63. Nonstationary fluctuation analysis was done after 10 min of
H2O2 reaction (Fig. 8, D-F).
During this period, we found a decrease in the number of channels by
about one-third with respect to the control value. The fitting
parameters are i = 34.6 ± 2.7 pA, N = 111 ± 8 channels, and Po = 0.6. After
30 min of H2O2 exposure, the variance vs. current plot in Fig. 8I does not reach a maximum, indicating
a clear decrease in Po. Because the variance vs.
current plot does not reach a maximum, it is not possible to fit the
data to Eq. 1. These experiments indicate that
H2O2 promotes both a decrease in the number of
active channels and in Po.

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Fig. 8.
Variance analysis of hSlo channel at different
times after H2O2 addition. A,
D, and G: mean current traces obtained from 256 traces recorded with the patch technique from a holding potential of 0 mV to a test pulse potential 120 mV at 0, 10, and 30 min after addition
of 8 mM H2O2 to the internal side,
respectively. B, E, and H: time course
of the variance. C, F, and I: variance
( 2) vs. mean current [I(t)]
fitted to the function: 2 =
iI(t) I(t)2/N (solid line),
where N is the number of channels and i the
unitary current. At t = 0 min, maximum
Po was 0.63, i = 37.0 ± 2.3 pA, and N was 155,700 ± 15; after 10 min of
H2O2 exposure, Po = 0.60, i = 34.6 ± 2.7 pA, and N = 111,000 ± 8. After 30 min of H2O2
exposure, a clear maximum cannot be observed, which implies that
Po 0.5. i was calculated from the
slope of the 2 vs. I(t) curve.
Internal [Ca2+] was 728 nM, and V is 120 mV.
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Effect of calcium after treatment with H2O2
on hSlo channel current.
At low [Ca2+], the addition of 18 mM
H2O2 produces a decrease in macroscopic current
after 20 min of exposure (Fig.
9A). This effect was reversed
by increasing the internal [Ca2+] to 100 µM. This
effect was similar to that observed in single-channel experiments (Fig.
3, C and F). H2O2
addition at the internal side induces a shift of the tail conductance
vs. voltage curves, with a clear decrease in the maximum conductance.
As can be observed in Fig. 9B, the effect of the oxidant can
be fully reversed by perfusing the internal side of the channel with a
H2O2-free solution containing 100 µM
Ca2+. In the presence of high Ca2+ (100 µM),
the addition of 18 mM H2O2 does not produce a
decrease in the macroscopic current after 20 min of exposure (Fig.
10, A and B).

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Fig. 9.
High [Ca2+] recovers the activity of
H2O2-modified channels. A:
macroscopic currents were recorded using the patch-clamp technique in
the inside-out configuration. The condition is the same as that in Fig.
7, but internal [Ca2+] = 498 nM and
[H2O2] = 18 mM. B:
Gtail vs. voltage curves for the traces shown in
A. Each point is the average of 5 patches in the inside-out
configuration. Gmax was 15,500, 6,000, and
14,800 pS for the Gtail vs. voltage curves taken
at 0, 20, and 30 min, respectively. [Ca2+] at
t = 30 min was 100 µM.
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|

View larger version (23K):
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Fig. 10.
High [Ca2+] supports activity of KV,Ca
against the deleterious effect of H2O2.
A: macroscopic current was recorded in the inside-out
configuration under the same conditions described for Fig. 7.
B: relative conductance
(G/Gmax = Po) vs. voltage curves. In the control condition
at t = 0, [Ca2+] was 100 µM
( ). , After 20 min of exposure to 18 mM
H2O2 addition. , After 40 min
of exposure to 18 mM H2O2. The fit curve was
the same that Fig. 7.
|
|
 |
DISCUSSION |
The interest in H2O2 as a biologically
active oxygen-derived intermediate is evident, because it is associated
to a series of alterations and effects in many different types
of cells. H2O2 is not by itself reactive enough
to oxidize organic molecules in an aqueous environment. Nevertheless,
H2O2 has the ability to generate highly
reactive hydroxyl free radicals through its interaction with
redox-active transitional metals (2). Hydroxyls result
from the decomposition of H2O2 via the Fenton
reaction and by interaction of superoxide with
H2O2 through the Haber-Weiss reaction
(41). The biological importance of
H2O2 stems from its participation in the
production of more reactive chemical species such as ·OH, and its
role as a source of free radicals has been emphasized rather than its
chemical reactivity. ·OH is considered one of the most potent
oxidants encountered in biological systems. However, because of its
extremely short half-life, it is effective only near the locus of its
production. The diffusion capability of ·OH is restricted to only
about two molecular diameters before it reacts with water
(41). Highly reactive ·OH readily react with a variety
of molecules, such as amino acids and lipids, by removing hydrogen or
by addition to unsaturated bonds (28). The lag time and
its dependence on [H2O2] can be explained
using a simple model in which n number of successful and
independent hits of the H2O2 with different
amino acid residues are necessary to "kill" a channel. A successful
hit is described by the irreversible reaction
|
|
where Xmod is the residue modified
by the hydrogen peroxide and k1 the second-order
rate constant describing the its chemical modification. In this model,
the time dependence of the probability of opening,
Po(t), is given by the expression
|
(4)
|
where
= 1/k1[H2O2] is the time
constant. The best fit to the data of Fig. 2B (4 and 8 H2O2 mM) was obtained with n = 103. This number is very large indeed and can be explained on the basis of the large number of cysteine residues (108) present in
the carboxy terminal of the KV,Ca channel. Below we show
that cysteine residues are the main target of the oxidant agent,
although it is likely that methionine or tryptophan residues are also
oxidized.1 Hovewer, other
mechanisms are possible, and the interpretation of the n
should be taken cautiously. The model proposed demands that the value
of 1/
must be directly proportional to the
[H2O2]. The inset of Fig. 2B shows
that the 1/
[H2O2] data are well
described by a straight line with a second-order rate constant
k1 = 0.56 s
1M
1.
The value obtained for k1 indicates that the
oxidation is extremely slow indeed, considering that the forward rate
constant in a diffusion-limited reaction is of the order of
108
109
s
1M
1. The very short half-life of the ·OH
can explain the low forward rate constant, k1,
for the channel oxidizing reaction (Fig. 2B), obtained using
the multiple-hit model because the effective [·OH] at the target
sites in the protein would be low. We note here that the oxidizing
reaction is a multistep process in which ·OH are formed via the
Fenton reaction, reacting afterwards with, for example, an
SH group
at a diffusion-controlled rate to form sulfinic or sulfonic acid
derivatives (see, e.g., 18, 36). Therefore, the effective [·OH]
should be in the nanomolar range to explain the low reaction rate found
in Fig. 2B.
In the present work we found that adding H2O2
to the cytoplasmic aspect of the KV,Ca channel produces a
decrease in its Po (Fig. 2). This effect was not
reversed by washing or after increasing [Ca2+] to 60 µM
(Fig. 3B). Therefore, H2O2 could be
involved in redox reactions with some vulnerable amino acid residues
(28). The protective effect showed by desferrioxamine and
cysteine before treatment with 23 mM H2O2 (Fig.
4) implies that the decrease in Po is mediated
by the ·OH generated by the Fenton reaction.
Of course, it is possible that the oxidant agent affects other
components associated to the membrane or to the channel; for example,
the target of the oxidizing agent could be an auxiliary
-subunit or
some membrane-bound enzyme able to promote channel phosphorylation. We
think that the bilayer experiments argue against that possibility,
because the skeletal muscle preparation does not contain
-subunits
and we are working in the absence of second messengers such as ATP or
cAMP. In what follows, we assume that the primary target of the ·OH
is the channel-forming protein.
The decrease in the KV,Ca channel activity by addition of
H2O2 to the intracellular side is highly
dependent on the oxidant concentration. Low
[H2O2] (8 mM) does not affect the
Po of KV,Ca channels in the first 8 min of a reaction. Afterwards, and in a very short time span,
Po values change drastically. Based on the
effect of desferrioxamine, the reduction in Po
can be attributed to the oxidizing action of the ·OH on free SH
residues of cysteines associated with the opening of the
KV,Ca channel. The differences in the effect of the
H2O2 when it is added to the intracellular or
extracellular side imply different access to essential targets. In
particular, oxidation of free SH residues of cysteines, present in
greater proportion at the intracellular side (27 amino acid residues
per subunit), could explain the observed difference. Twenty-four of
these residues occur in transmembrane or intracellular domains and are
largely concentrated in the carboxy terminus of the hSlo
protein. We think that the external cysteines (C14, C141, and C277) do
not play an important functional role in determining Po, because their replacement by serine produces
channels indistinguishable from the wild-type in terms of
voltage-Ca2+ dependence and H2O2
sensitivity (data not shown).
To determine whether the Po decrease of the
KV,Ca channel by ·OH could be attributed to the oxidation
of free SH residues of cysteine, the effect of
H2O2 was compared with that of DTNB. This is a
hydrophilic agent specific for the oxidation of free SH groups in
proteins. The presence of 2 mM DTNB decreased the Po value to an extent similar to that observed
with H2O2. This effect was reversed by addition
of 2 mM DTT (Fig. 6A). On the other hand, the addition of 2 mM DTT to a channel previously treated with
H2O2 only produces a partial recovery in the
Po (Fig. 5B). These results would
indicate that KV,Ca channel of rat skeletal muscle can be
regulated by compounds that alter the redox states of sulfhydryl
groups. Similar behavior was described for a smooth muscle
KV,Ca channel (38, 39) and a
voltage-insensitive K(Ca2+) channel of intermediate
conductance present in bovine aortic endothelial cells
(5). Cai and Sauvé (5) show that the oxidative effects of H2O2 were observed at
H2O2 concentrations ranging from 0.5 to 10 mM.
The oxidative effect of H2O2 was similar to
hydrophilic oxidative reagents such as DTNB. The difference observed
between the KV,Ca channel activity recovery by DTT in pretreated samples with H2O2 (Fig.
5C) (recovery 57%) and that with DTNB (Fig. 6A)
(recovery 85%) indicates that the ·OH could have a more generalized
oxidative effect than DTNB. Besides cysteines, other amino acids such
as tryptophan and/or methionine can be the target of the ·OH
(7, 23, 28).
We note here that the addition of H2O2 in
presence of high calcium (Fig. 9) does not produce a macroscopic
conductance decrease after 20 min of exposure. In this condition, high
internal [Ca2+] would act as a protective
Ca2+ agent for the different sensitive groups exposed in
the carboxy-terminal region, particularly at calcium binding sites
(29). These experimental facts could be
important, due to the fact that the intracellular Ca2+ is
modulating and potentially protecting groups that can be oxidized and
are associated to the opening and closing of the KV,Ca channel.
Oxidation effects depend on the type of
K+ channels.
The effect of ROS on the KV,Ca channel differs from that
reported for voltage-dependent K+ or the human
ether-à-gogo-related gene (HERG) channels. For example,
t-butyl hydroperoxide reversibly increases the activity of
both Kv1.4 and Kv3.4. This effect was attributed to an attenuation or
removal of the fast inactivation processes (11).
Enhancement of ROS production induced by the perfusion with
Fe2+ and ascorbic acid caused an increase in HERG outward
K+ currents (34). On the other hand, a
decrease in ROS levels achieved by perfusion with ROS scavengers
inhibited the resting outward currents induced by HERG channels and
prevented their increase induced by ROS. Rose bengal (generator of
singlet oxygen, 1O2) produced a decrease of
channel activity in the case of Shaker, Kv1.3, Kv1.4, Kv1.5, and Kv3.4
channels expressed in Xenopus oocytes. Duprat et al.
(11) argued that these observations might be important in
disease states. Kv1.4 and Kv1.5 are fast inactivating channels expressed in cardiac cells, and their inhibition by ROS can contribute to the major electrophysiological disorders that occur during reperfusion-induced arrhythmias after ischemia and during heart failure induced by chronic pressure overload (3). Evidence of a direct effect of H2O2 on ATP-sensitive
K+ (KATP) channels was inferred from studies
where ROS effects were examined on excised membrane patches. Ichinari
et al. (16) observed a dose-dependent
H2O2-induced increase in
Po of the KATP channel. H2O2-induced irreversible inhibition of the
activity of KATP channel in skeletal muscle has been
attributed to inhibition via oxidation of SH groups (40).
On the other hand, sarcoplasmic reticulum Ca2+ release
channel (ryanodine receptor) is differentially affected by different
ROS. Singlet oxygen causes an irreversible damage of the ryanodine from
cardiac muscle after a brief transient period of activation
(15). However, 5 mM H2O2 activates
the same channel even at 0.45 nM cytosolic calcium, a condition in
which the channel is normally silent. The activation occurs abruptly
after a lag period of a few minutes (4). The activating
effect of H2O2 has also been found for the
ryanodine receptor from skeletal muscle channel of the rabbit and frog
(12, 26). The rabbit channel is somewhat more sensitive;
it becomes activated at 0.1 mM, and in this preparation 1-3 mM
H2O2 inhibit channel activity
(12).
The range of [H2O2] used by different authors
varies from 0.1 to 50 mM (19). The exact physiologically
significant concentration is not clearly defined and may depend on the
cellular type. For example, the potassium channel KShIIID.1 expressed
in Xenopus oocytes is sensitive to 10 µM
H2O2. The current through this particular channel is very similar to the currents sensitive to the arterial O2 pressure found in chemoreceptor neurons, where 10 µM
H2O2 does modify neuronal activity
(37). The intracellular [H2O2]
reached during exercise in skeletal muscle have not been determined,
but because H2O2 effects develop progressively
after repeated tetanic contractions, the accumulation of
H2O2 could affect skeletal muscle channels.
Recently, Tang et al. (35) found that at the intracellular
side, methionine oxidation by chloramine-T produces an increase of the
Po mediated by an increase in voltage-dependent
opening transitions and a slowing down of the closing transition rate.
They observed that the stimulatory effect of chloramine-T is maintained
in the cysteine-less mutant channel (35). Our results indicate that the ·OH had a wide oxidative effect, which does not
contradict their results.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Eduardo Rosenmann for critical reading of the
manuscript, Dr. Osvaldo Alvarez for suggesting to us the multiple-hit model, and Luisa Soto for excellent technical assistance. Drs. Enrico
Stefani and Ning Zhu kindly shared with us the external cysteine-less mutant.
 |
FOOTNOTES |
This work was supported by Chilean Fondo Nacional de Investigacion
Científica y Tecnológica Grants 398-0005 (M. Soto),
100-0890 (R. Latorre), and 198-1053 (C. Vergara); by Cátedra
Presidencial en Ciencia (R. Latorre and E. Lissi); and the Human
Frontier in Science Program (R. Latorre). Centro de Estudios
Científicos is a Millenium Science Institute.
1
We show below that ·OH via the Fenton reaction
mediate the H2O2 effect. This fact will not
affect the conclusion extracted using the model used to fit the
Po-time data since the [ · OH] is directly
proportional to the [H2O2].
Address for reprint requests and other correspondence: M. A. Soto Arriaza, Centro de Estudios Científicos (CECS), Av.
Arturo Prat 514, Valdivia, Chile (E-mail: marcos{at}cecs.cl).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00167.2001
Received 2 April 2001; accepted in final form 17 October 2001.
 |
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