© 1996 American Heart Association, Inc.
Articles |
Cell DistensionInduced Increase of the Delayed Rectifier K+ Current in Guinea Pig Ventricular Myocytes
From the Department of Physiology, Faculty of Medicine, Kyoto (Japan) University.
Correspondence to Dr T. Mitsuiye, Department of Physiology, Faculty of Medicine, Kyoto University, Sakyo-Ku, Kyoto 606, Japan.
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Abstract |
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Abstract Single ventricular myocytes of guinea pig heart were distended by applying a positive pressure of 5 to 20 mm Hg in the pipette during the whole-cell voltage clamp. The amplitude of delayed rectifier K+ current (IK) was increased by
1.5 times, whereas the inward rectifier
K+ current was scarcely affected. The increase of
IK was reversible by applying a negative pressure of
-10 to -30 mm Hg accompanied by shrinkage of the inflated
cell. This response of IK was largely attributed to the
E-4031–insensitive component of IK. The fully
activated current amplitude, measured using long-lasting
depolarizing pulses (>30 seconds) to +60 mV, was increased by the cell
distension. The activation time course of IK during the
long pulse consisted of more than three exponential components, and the
slowest time constant was decreased by the distension from control
20.2±7.7 seconds (n=4) to 7.6±1.6 seconds (n=5).
We failed to detect
an involvement of microtubules or microfilaments, protein kinase C, and
Ca2+ in the inflation-mediated increase of
IK.
Key Words: delayed rectifier K+ current • ventricular myocytes • guinea pig hearts • membrane stretch • cytoskeleton
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Introduction |
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Depolarization of the ventricular cell membrane immediately shuts off the inward rectifier K+ channels, providing the low background membrane conductance for generation of the long-lasting plateau of the action potential. Continuous depolarization then gradually increases the K+ conductance (IK). It is generally considered that the activation of IK is mainly responsible for gradual repolarization during the plateau, and finally, the outward current through the inward rectifier K+ channel terminates the ventricular action potential. The amplitude of IK is modulated by various factors, such as stimulation of the ß-adrenergic receptors,1 2 phorbol ester stimulation,3 4 and intracellular Ca2+.5
Recently, whole-cell voltage-clamp experiments revealed that the magnitude of IK is dramatically changed by altering the osmolarity of the bathing solution.6 7 Exposure to a 70% hypotonic solution almost doubles the magnitude of IK, whereas a 130% hypertonic solution halves it. This response of IK was consistently observed and appeared earlier than the delayed increase of the swelling-activated Cl- conductance.7 Superfusion of myocytes with hypotonic or hypertonic solution may increase or decrease cell volume, respectively, and the changes in the cell volume may alter the tension applied to the sarcolemma. However, our previous study6 failed to observe changes in IK during mechanical stretch applied along the longitudinal axis of the ventricular myocytes. The present study demonstrates that the increase of IK similar to that induced by the hypotonic superfusion is seen when a pipette solution is injected directly into the cell. It is suggested that the number of available IK channels is increased by the membrane stretch in a reversible manner. However, the mechanism of this effect remains unresolved and does not seem to involve PKC, Ca2+, or the cytoskeleton.
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Materials and Methods |
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Single-Cell Preparation
Single ventricular cells were isolated from guinea pig (300 to 400 g body weight) hearts using essentially the same enzymatic dissociation technique as described previously.8 9 In brief, each guinea pig was deeply anesthetized with pentobarbital sodium (30 mg/kg IP), and the heart was dissected out and perfused for 20 minutes with a Ca2+-free Tyrode's solution containing 0.04% collagenase (type I, Sigma Chemical Co) using the Langendorff perfusion method. After the enzymatic treatment, single myocytes were dispersed from the left ventricle in a high-K+ low-Cl- solution and stored in the cell culture medium (Eagle's medium, Flow Laboratories) at room temperature (24°C to 26°C) for later use.
Solutions
The composition of the control Tyrode's
solution was (mmol/L)
NaCl 140.0, KCl 5.4, CaCl2 1.8, MgCl2 0.5,
NaH2PO4 0.3, glucose 5.5, and HEPES 5.0, and
the pH was adjusted to 7.4 with NaOH. CaCl2 was omitted if
necessary. In some experiments, a hypotonic (Figs 5B
and
7) or a
hypertonic (Fig 5C) solution was prepared as described
previously.7 In brief, the hypotonic solution (70%
osmolarity) was prepared by decreasing the concentration of NaCl to 100
mmol/L in the control Tyrode's solution, and the hypertonic solution
(130% osmolarity) was prepared by adding 90 mmol/L mannitol to the
control Tyrode's solution. An isotonic solution was also prepared by
adding 80 mmol/L mannitol to the hypotonic solution. As noted
previously,7 no significant difference was noticed in
IK between the two isotonic solutions (Tyrode's solution
and hypotonic solution+mannitol). The L-type Ca2+ current
was blocked by adding 2 mmol/L NiCl2 to all bath solutions.
The bath solution was exchanged by switching the perfusates at
the inlet of the chamber. In all experiments, the temperature of the
bath solution was kept at 35±0.5°C.
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Colchicine, cytochalasin B and D, and vinblastine sulfate (Sigma) were used to disrupt the cytoskeleton. Different doses of those agents were tested in the present study compared with previous studies.10 11 12 Staurosporine (Wako) was used to inhibit PKC. These chemicals were prepared freshly before the experiment. Cytochalasin B and D and staurosporine were dissolved in dimethyl sulfoxide and vinblastine sulfate was dissolved in acetone before addition to the external solutions. The final concentration of dimethyl sulfoxide or acetone in the salines was <0.1%. Experiments with cytochalasin B and D and vinblastine sulfate were performed in relative darkness to avoid photodestruction. E-4031 was kindly provided by Eisai Pharmaceutical Co and was dissolved in distilled water to make 10 mmol/L stock solution.
The patch pipettes were filled with an internal solution containing (mmol/L) potassium aspartate 140, MgCl2 5.0, K2-ATP 5.0, Na2-phosphocreatine 5.0, EGTA 10, and HEPES 5.0 (pH 7.2 with KOH).
Whole-Cell Current Recording
Single ventricular cells were
voltage-clamped
using the whole-cell configuration of the patch-clamp
technique13 (patch-clamp amplifier, Axopatch-lD,
Foster). The resistance of the pipette was 2.5 to 4.0 M
when filled
with the internal solution. The membrane current and potential were
recorded on a digital magnetic tape (RD-101, TEAC) for later
computer analysis (PC98, NEC). The cell membrane capacitance
was calculated by integrating current recorded by 10-mV
hyperpolarizing steps from a holding potential of -40 mV. The
liquid junction potential of -10 mV between the pipette solution
and the external solution was corrected for all membrane potential
recordings.
The changes of the ventricular cell volume during the osmotic cell swelling or shrinkage have been approximated by geometric measurements14 15 and were compared with the activation of a volume-sensitive Cl- channel.16 According to these previous studies, we measured the cell area under the microscope as an approximation of the cell volume. The image of the single ventricular cell under the whole-cell voltage clamp was recorded with a video recorder, and the single cell area was measured on the video screen. The cell was inflated or deflated by applying a positive or negative pressure into the pipette, which was monitored by a pressure transducer (Spectramed).
Data are given as mean±SEM. Statistical significance was determined using Student's paired t test.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Increase of IK by Mechanical Inflation of the Ventricular Cell
The ventricular myocytes were slowly inflated by applying a positive pressure of 5 to 20 mm Hg into the glass pipette during the whole-cell voltage clamp. In the experiment shown in Fig 1A
and 1B, when a positive pressure of 5 mm Hg
was
applied into the pipette, the amplitude of IK slowly but
continuously increased, accompanied by an enlargement of the cell area
observed under the microscope. We failed to observe the activation of a
volume-sensitive Cl- current, which was typically
represented by an increase of instantaneous current jump at
the pulse onset in the previous hypotonic cell swelling.7
The amplitude of IK usually remained constant or slightly
decreased after the release of the positive pressure and was decreased
by applying a negative pressure. The recovery of IK was
accelerated by increasing the negative pressure as shown in Fig
1A.
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, 0 mm Hg), after cell inflation (
, 5
mm Hg), and after deflation (
, -20 mm Hg), obtained at
times corresponding to those in panel A. C, Current-voltage
relations measured at the onset of pulse (left) and near the end of
pulse (right). Different symbols indicate the corresponding cell
conditions in panels A and B.
The current-voltage relations were measured when the
current change
reached a steady state in each experimental condition. The original
current recordings in Fig 1B
indicate that both IK
during depolarization and the tail current on repolarization were
increased by the mechanical cell distension. No obvious change was
noticed in the double exponential time course of activation or
deactivation of IK as examined by the semilogarithmic plot
of the time-dependent current.7 Namely, the fast and
slow time constants of activation were 106±5 and 708±24
milliseconds
in the control condition and 96±8 and 739±19 milliseconds after
the
inflation; those for deactivation were 95±7 and 310±25
milliseconds
in the control condition and 94±9 and 290±27 milliseconds after
the
inflation (n=7).
In Fig 1C, the isochronal
current-voltage relations measured at
the onset of the pulse (left) showed no significant difference among
the control (circles), inflated (squares), and recovery (triangles)
values, suggesting that the inward rectifier K+ current was
not affected by the inflation. The slope conductance between -70
and -90 mV as a measure of the inward rectifier K+
conductance was 104±12% (n=10) of the control value in the
inflated
cells. However, in the current-voltage relations measured near the
end of the pulse (Fig 1C, right), the outward current was
markedly
increased at potentials more positive than -10 mV by the cell
distension. It seems that the voltage range of activation of
IK was not changed. Essentially the same findings were
obtained in seven experiments. The amplitudes of the outward current
measured near the end of the pulse and the tail current were 2.26±0.29
and 0.73±0.09 nA (n=7) in the control cells, respectively; those
in
the inflated cells were increased to 152±9% and 148±8%
(n=7) of the
control values, respectively. The input capacitances of the cell,
measured before and after the inflation, were not significantly
different (P>.1). The capacitance was 212±23 pF in the
control condition and 196±16 pF (n=7) after the cell distension.
A small outward shift of the holding current was observed in 6 inflated cells, and the amplitude of shift ranged between 0.10 and 0.25 nA (0.16±0.03 nA). The mechanisms of this shift were not examined in the present study. In 3 of 10 experiments, we failed to observe any change in IK or in the cell shape, and increasing pressure above 30 mm Hg simply destroyed the tight seal at the pipette tip. The positive pressure used for the inflation of the cell was between 5 and 20 mm Hg, and the negative pressure for the deflation was -10 to -30 mm Hg.
The E-4031–Insensitive Component Is Responsible for the
IK Response
IK has been separated into two
components on the basis
of their sensitivity to some K+ channel blocking compounds
such as E-4031. There is a rapidly activated drug-sensitive
component (IKr) and a slowly activated
drug-insensitive component (IKs) in guinea pig
ventricular myocytes.17 In Fig 2, the whole-cell
current in response to a
depolarizing pulse to +10 mV was recorded with and without E-4031.
In the control condition, the amplitude of IK was slightly
decreased by 5 µmol/L E-4031 (Fig 2A), and the
drug-sensitive
component (difference current, IKr) showed a relatively
rapid rise and decay on depolarization and repolarization,
respectively. The relative amplitude of E-4031–sensitive current was
measured from IK,tail on repolarization and was 31±10% of
the control value (n=4). Under the effect of the drug, however,
IK was still enhanced by the mechanical cell distension
(201±17% of the control value, n=3).
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indicates the control condition; 
, in the presence of the
drug. In panel B, 
,
during cell inflation by the application of 7 mm Hg pressure into the
recording pipette; and 
, in the presence of the drug. The
zero current level is indicated by short lines at the beginning of the
trace. The difference currents at the bottom were obtained by the
subtraction indicated.
In a separate series
of experiments, E-4031 was applied only after
IK was enhanced by the cell inflation. The drug decreased
the enhanced tail amplitude by 10.5% in the experiment shown in Fig
2B. In eight cells, IK,tail was increased to
177±10% of
the control value, and the drug-sensitive component amounted to
23±3% (n=8).
Effects of E-4031 on IK were also examined in the hypotonic cell swelling (70% osmolarity; data not shown). As in the case of the mechanical cell distension, IK was enhanced by the hypotonic cell swelling even under the effect of E-4031. This finding is in agreement with the recent report by Rees et al,18 who reported that osmotic cell swelling significantly increased IKs. On the basis of the above experiments, we conclude that the increase of IK in inflated ventricular cells is largely due to the drug-insensitive component.
IK During a Continuous Depolarization
When
IK was enhanced by the hypotonic
solution,7 no obvious change was observed in the
double-exponential time course of activation and deactivation or in
the voltage dependence of activation if the currents induced by
depolarizing pulses of several seconds were analyzed by
assuming a steady current level by a best-fit
routine.19 However, it is essential to determine the
steady state current level to determine the activation kinetics in
detail. So we made an attempt to record the fully activated
amplitude of IK by prolonging the depolarizing pulse.
The
current recordings induced by depolarizing pulses of
different durations are shown in Fig 3A. The outward
current did not reach steady state even using a 32-second
depolarization. The extremely slow late current change induced by the
continuous depolarization might be due to unknown current systems,
which might overlap on the relatively fast activation of
IK. For example, [Ca2+]i may
increase during depolarization accompanied with a gradual activation of
the Ca2+-dependent Cl-
current.20 21 22 This might cause an
increase of the outward
current but not the tail current at -40 mV, which is near the
Cl- equilibrium potential. To test the possibility of
overlapping currents, we examined the relationship between the
amplitude of the outward current (IK) during the
depolarization and that of IK,tail. The ratio of
IK,tail to IK was plotted against the duration
of the depolarizing pulse on a logarithmic time scale (Fig 3).
Although
the ratio of IK,tail to IK was larger for short
depolarizing pulses (0.60±0.04, n=6 for 125-millisecond pulse)
as
reported previously,17 it became quite constant for the
pulses >1 second. The value of 0.36 was almost equal to the ratio of
the driving forces for K+ at +50 mV and -40 mV.
This
finding may support the view that the extremely slow activation of the
outward current is due to IK.
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t) of the depolarizing pulse
to +50 mV was varied from 125 milliseconds to 32 seconds, and the
current recordings were superimposed. B, The ratio of
IK,tail/IK was determined for each
duration of the pulse and plotted against the pulse duration (
The membrane potential, which
saturates the steady state
activation of IK, was determined in Fig 4. A pulse
duration of 30 seconds was used, and the
amplitude of IK,tail at -40 mV was measured. The
relationship between the amplitude of the tail current and the test
potential was fitted with a Boltzmann curve. The half saturation was at
26.8±3.5 mV, and the slope factor was 16.1±1.2 mV (n=4).
These values
are different from those determined previously using relatively short
pulses.7 17
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Fully Activated Amplitude of IK During the
Cell Distension
IK was recorded by depolarization to
+60 mV, where
activation is almost complete (Fig 4), before and during the
mechanical
inflation of ventricular cells. In the experiment shown in
Fig 5A, the gradual enhancement of IK by
applying a 10 mm Hg pressure was monitored by repeating the usual
2-second depolarizing pulses to +60 mV, and the long-lasting
depolarizations were applied as required. It was evident that the
saturating amplitude of IK was increased by the cell
distension. In four experiments using 5 to 20 mm Hg positive pressure,
the saturating amplitude of IK during depolarization was
increased to 170±30% of the control value. This finding suggests that
the inflation of the ventricular myocyte increases the
number of available IK channels.
The increase of fully
activated IK induced by the
cell distension was also confirmed during a continuous depolarization.
Because of the variable time lag for the development of
IK after applying the positive pressure into the pipette,
we used the osmotic method. In Fig 5B, hypotonic solution was
applied
under a continuous depolarization to +60 mV after saturating
IK activation in the control solution. It is evident that
IK was further increased by the hypotonic inflation. After
terminating the continuous depolarization, further increase of
IK in the hypotonic solution was confirmed by repeating the
2-second depolarization. Accordingly, the saturating level of
IK in the hypotonic solution was further increased as
revealed by the second continuous depolarization. During this
depolarization, switching back to the isotonic solution almost
completely reversed the increase of IK. Thus, in three
cells, the fully activated amplitude of IK was
139±7% of the control value in the hypotonic solution.
The
examination of the fully activated amplitude of
IK was further extended to the hypertonic solution (Fig
5C). The superfusion of hypertonic (130% osmolarity) solution
decreased the fully activated current level in a reproducible
manner. On the average, the saturating current level of IK
decreased to 65±7% (n=5) of the control value in the hypertonic
solution.
Kinetic Properties of IK Examined by the
Long-Lasting Depolarization
Changes in the time courses of
IK activation and
deactivation by the cell inflation were examined using the
long-lasting depolarizing pulse. Fig 6A shows the
semilogarithmic plot of the time-dependent component of
IK during the pulse to +60 mV before (left) and after
(right) the mechanical cell inflation by applying 10 mm Hg. It is
evident that the initial rapid increase of IK within 2
seconds is followed by a very slow exponential activation phase. By
fitting a line to the late current change during depolarization, the
exponential time constant of the slowest current component was
determined to be 20.2±7.7 seconds (n=4) in control cells and
7.6±1.6
seconds (n=5) in inflated cells. This kinetic change may be
supplemented by measuring the steady state activation. However, such
measurements using long-lasting pulses before and after the cell
inflation were not feasible and accurate because of spontaneous current
fluctuations during the extremely long experimental time. In three
experiments, a trend of negative shift of the half-activation
voltage was suggested; namely, it was shifted from 23.9, 35.4, and 28.9
mV to 16.3, 11.9, and 13.3 mV after the cell inflation,
respectively.
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The IK,tail recorded on repolarization to a
holding
potential of -40 mV was fitted by a sum of two exponentials (Fig
6B), as in the previous study, using a short (<3-second)
conditioning
depolarization.7 The deactivation time course was not
significantly changed; the fast and slow time constants of the tail
were 138±19 and 457±93 milliseconds, respectively, in the
control
state and 134±16 and 415±77 milliseconds, respectively, in the
inflated state (n=3).
Taken together, we conclude that only the
slowest (
>5-second) phase
of activation of IK during the extremely long pulse is
markedly accelerated by the cell distension, whereas the fast
double-exponential time course of both activation and deactivation
remains unchanged.
Effects of Disrupting Cytoskeleton
It has been postulated
that a viscoelastic cytoskeletal network is
essential for the regulation of the stretch-activated
channels.23 24 In the present study, the involvement
of microfilaments or microtubules in the distension-induced
modulation of IK was tested by disrupting the cytoskeletal
elements by directly introducing cytoskeletal modifiers into the
cytoplasm through the patch pipette. Myocytes were dialyzed with an
internal solution including cytochalasin B (2 µmol/L). In order to
ensure the equilibration of the pipette solution with intracellular
medium and the steady effect of cytochalasin B, the effect of the cell
distension was examined at least 7 minutes after the whole-cell
current recording was started. In a series of four experiments,
IK recorded with a 2-second depolarizing pulse was
increased by inflating cells (5 to 10 mm Hg positive pressure), which
was reversed by applying a negative pressure. The amplitudes of
the outward current and IK,tail during the cell inflation
were 160±9% and 150±11% (n=4) of the control value,
respectively.
No obvious change was noticed in the time course of IK
enhancement during the application of positive pressure. The response
of IK to cell distension was also observed when 50 nmol/L
(n=3) and 2 µmol/L vinblastine sulfate (n=2) or 2 µmol/L
colchicine
(n=4) was added in the pipette solution to disrupt microtubules.
To further confirm that neither the microfilaments nor the microtubules are involved in the IK response to the cell distension, ventricular cells were incubated for 3 to 5 hours in cell culture medium containing cytoskeletal modifiers. Then the whole-cell clamp was carried out in the continuous presence of the agent under investigation. Agents used were cytochalasin B (10 µmol/L, n=3; 100 µmol/L, n=2), cytochalasin D (10 µmol/L, n=3; 50 µmol/L, n=2), vinblastine sulfate (10 µmol/L, n=3), and colchicine (0.1 mmol/L, n=3; 0.2 mmol/L, n=4). None of these agents significantly affected the reversible increase of IK by the cell distension. Similarly, the hypotonic solution increased IK in a reversible manner under these conditions.
Other Putative Mechanisms for the Distension-Induced Modulation of
IK
An increase of
[Ca2+]i or protein
kinases was ruled out as a mechanism of the IK response to
hypotonic cell inflation in a previous study.7 Since
inflation of the ventricular cells was achieved by
injecting an internal solution containing 10 mmol/L EGTA in the
present study, an increase of [Ca2+]i is
unlikely to mediate the observed response. Confirming this view, the
IK response was also observed when an elevation of
[Ca2+]i was prevented by the use of a
nominally Ca2+-free external solution. The amplitude of
IK at the end of a 1-second depolarizing pulse to +60 mV
and the amplitude of IK,tail were increased to 175±13%
and 183±28%, respectively, of the control amplitude (n=3). The
increase of IK was also observed in the inflated cells when
2 µmol/L staurosporine, a specific inhibitor
of PKC, was included in the external solution. The amplitudes of the
outward current and tail current of IK were increased to
143±8% and 130±7%, respectively, of the control amplitude
(n=7).
Thus, we conclude that the IK response to the mechanical
cell inflation is not mediated by an elevation of
[Ca2+]i or PKC.
Increase of IK Parallel to the Osmotic Swelling of the
Ventricular Cell
The inflation of the cell was always observed when
IK
was increased by applying positive pressure into the pipette. However,
the increase of cell area was not always uniform over the entire cell,
probably because of the point source of the fluid injection. Therefore,
the relation between the cell swelling and the amplitude of
IK was compared using the hypotonic cell swelling method,
which caused homogeneous cell inflation. In Fig 7A, the
increase of IK as examined by
2-second depolarizing pulses (upper panel) is compared with the
normalized cell area (lower panel) plotted on the same time scale. Both
IK and the cell area were increased with a similar time
course to reach a new plateau level after the hypotonic superfusion,
and they were reversed by the superfusion of the control solution. The
half-times for the enhancement and recovery of IK were
48.6±6.3 seconds (n=7) and 41.0±2.5 seconds (n=5);
those for the cell
area were 61.4±6.0 seconds (n=7) and 45.0±9.6 seconds
(n=4),
respectively.
The relative amplitude of IK was plotted
against the
relative cell area in Fig 7B (n=6), which indicates that
the increase
of IK was nearly proportional to the increase of the cell
area. However, in one experiment the increase of the current was much
delayed compared with that of the cell area.
Shortening of the Action Potential by Mechanical
Inflation
In our previous study, we concluded that IK takes
the
role of osmoelectrical signal transduction.7 The role of
IK in the mechanotransduction is clearly shown in Fig
8A, where the action potential was recorded before
(a) and after (b) the mechanical cell inflation. Inflating the cell
shortened APD50 from 340 to 238 milliseconds and
APD90 from 365 to 250 milliseconds. The time derivative of
the membrane potential is shown in Fig 8B, which suggested that
the
reduction of action potential is due to the increased density of the
outward current during phase 2 and 3 repolarization. In five
experiments, mean APD50 was decreased to 68±4% and
APD90 was decreased to 69±4% of the control value. These
observations are consistent with the enhancement of
IK during cell inflation.
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Discussion |
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Mechanisms of the IK Response
The increase of IK induced by the mechanical cell inflation in the present study is quite comparable to that induced by the hypotonic cell swelling in the previous studies.6 7 18 In both cases, the increase of IK was reversible, and the gating kinetics remained almost constant after the cell swelling, except for the very slow activation that was revealed using a pulse longer than 30 seconds in the present study. The increase of IK during mechanical cell inflation was also observed when an elevation of [Ca2+]i was prevented by the use of Ca2+-free internal and external solutions or by inhibiting PKC with staurosporine, excluding Ca2+-mediated mechanisms from the signal transduction.6 Furthermore, the present study demonstrated an almost linear relationship between the cell surface area and the magnitude of IK during the course of the osmotic cell swelling (Fig 7
). Thus, these
findings
strongly suggest a direct coupling between the activity of the
IK channels and the membrane stretch irrespective of
osmotic or mechanical cell inflation. It should be noted that the
present findings using the cell distension technique are free from
complications of dilution or loss of the intracellular components,
which remained to be examined in the previous studies of the osmotic
cell swelling.26 27 The mechanical cell distension of the myocyte induced by injecting pipette solution consistently caused an increase of IK. However, IK was not significantly changed when the cardiac myocyte was stretched along its longitudinal axis.6 This difference might be explained by different deformations of the cell membrane. In the latter case, the diameter of the elongated cell might be decreased by the cell extension to maintain a constant cell volume, whereas the cell distension might cause a two-dimensional stretch on the cell membrane, which might be essential for the IK response, as suggested in the stretch-activated ion channels.23 28
The present study failed to observe any modulation of the IK response to the mechanical inflation after treating myocytes with vinblastine, colchicine, or cytochalasin, excluding the involvement of the microtubules or microfilaments. The involvement of the cytoskeletal elements for the channel activity has been examined in a variety of cells and tissues, and the contribution of microtubules was consistently excluded.11 24 On the other hand, a wide variety of effects have been reported when microfilaments were destructed: an inhibitory effect on the IKs channel expressed in oocytes,10 no effect on the swelling induced Cl- current,29 and a stimulatory effect on the stretch-activated channel.11 23 The inhibition of the channel response may suggest an essential role of the microfilaments in the transduction of the membrane mechanical stress to the channel activity, whereas the enhanced channel activity may suggest that stretching the membrane was more effective when the cytoplasmic viscoelasticity was eliminated by the disruption of the microfilaments. It should be noted, however, that the present findings do not completely exclude the role of the cytoskeletal elements other than the microfilaments and microtubules. Sokabe et al30 suspected that spectrin/dystrophin submembrane cytoskeleton might be the tension-bearing element in stretch-activated channels because the lipid membrane was stress free and the destruction of microfilaments does not abolish the increases of the channel activity.
Busch et al10 expressed K+ channels by injecting cRNA from rat kidney in Xenopus oocytes and recorded IKs, which showed voltage-dependent activation similar to IK in the cardiac muscle. Interestingly, the amplitude of IKs increased in response to the hypotonic superfusion of the oocyte, and this response was present even in the presence of the protein kinase inhibitors. However, different from the cardiac IK, the hypotonic increase of IKs was suppressed by removal of Ca2+ in the external solution and by treatment of oocytes with cytochalasin D. These authors suggested that increase in [Ca2+]i changes the cytoskeletal organization of oocytes, thereby increasing IKs.
Stretch-Induced State Transitions of the IK
Channel
In guinea pig ventricular cells, the amplitude of the
E-4031–sensitive component of IK was relatively small (Fig
2), and the increase of IK by cell inflation was
largely
due to the drug-insensitive component. The amplitude of the
drug-insensitive component of IK (I) is
described as follows:
 |
where N is the total number of available channels, i the unit amplitude of the single-channel current, and Po the open probability of the channel. Our noise analysis7 suggested that i was not modified during the IK increase in the hypotonic cell swelling. Thus, the increase in I is attributable to an increase in NPo. However, a simple increase of N due to an increase of membrane area (eg, the fusion of intracellular membrane vesicles to the surface membrane) is difficult to assume, since the membrane capacitance remained constant after the cell distension, as in the case of the hypotonic cell inflation. A model that we use to explain our experimental findings includes an unavailable (resting) mode and an available (active) mode (see also the stretch-activated channel model proposed by Sachs24 ). The transition between these two modes may be determined by both the membrane potential and the membrane stretch:
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The active
mode includes several closed states and an open state.
This model assumes that more than half of the IK channels
are in the active mode in the isotonic solution and that the rest of
the channel population is in the unavailable resting mode. The gating
kinetics in the active mode (Po) is assumed not to
be affected by the cell distension, according to the experimental
finding that the voltage-dependent gating kinetics remained the
same as far as IK during short depolarizing pulses and
IK,tail are concerned. The slowest activation process
during the continuous depolarization (Fig 6A) is attributed to
the
recruitment of the unavailable channels through a voltage-dependent
transition from the resting mode to the active mode. The cell inflation
may shift equilibrium to the right between the resting and active
modes, probably by increasing the forward rate constant. The increase
in the number of functional IK channels due to an
aggregation of subunits into a channel was proposed in the
IKs channels expressed in Xenopus
oocytes.31 The hypothesized transition from the resting
mode to the active mode in the present study should also include
this aggregation mechanism if applicable to the IK
response. Unfortunately, the quantitative comparison of the present
model with experimental recordings was not practical because of
the difficulty in analyzing the unusually slow activation kinetics of
IK.
Action Potential Configuration
The duration of the action
potential was shortened by the
mechanical cell inflation in the present study (Fig 8). This
finding is in good agreement with the shortening of the action
potential in the whole-heart preparation, which was overloaded by
the intraventricular balloon method.32
The increase in ventricular volume might cause a
two-dimensional stretch of the membrane. The amplitude of the net
current calculated from the time derivative of the action potential was
not obviously changed by the cell inflation during the plateau, whereas
IK under the voltage clamp increased by 1.5-fold. The
increase of the net outward current by cell inflation was evident only
during the rapid repolarization (phase 3). To explain this apparent
discrepancy, the activation of IK was reconstructed by
using the IK model described by Kiyosue et
al25 along the time course of the recorded action
potential as shown in Fig 8C. When the limited conductance of
IK was increased by 1.5-fold in the model calculation, the
amplitude of IK was increased only by 1.05 times at 150
milliseconds after the onset of the action potential during cell
inflation. This is because the negative shift of the plateau potential
level decreased Po of the IK channel; eg, the
Po at 150 milliseconds was 0.27 for the control condition
and 0.22 for the depressed action potential in the model calculation.
However, the model calculation clearly indicated that the increase of
IK is primarily responsible for the shortening of the
action potential. The outward current during the rapid repolarizing
phase (Fig 8B) was increased when the action potential was
shortened.
At present, we cannot specify the underlying membrane current
change for this increase of the net outward current.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a scientific research grant from the Ministry of Education, Science, and Culture of Japan. Dr Wang was also supported by the Kato Asao International Scholarship Foundation. The authors thank Dr K. Manabe for his discussion, Dr Sian A. Rees for reading the manuscript and discussion, M. Fukao for his technical assistance, and K. Fujida for her secretarial work.
Received May 30, 1995; accepted December 1, 1995.
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References |
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