© 1995 American Heart Association, Inc.
Articles |
Effects of Delayed Rectifier Current Blockade by E-4031 on Impulse Generation in Single Sinoatrial Nodal Myocytes of the Rabbit
From the Department of Physiology, University of Amsterdam (Netherlands), Academic Medical Centre.
Correspondence to E. Etienne Verheijck, Department of Physiology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, Netherlands.
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Abstract |
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Abstract The role of the delayed rectifier current (IK) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit. We used the class III antiarrhythmic drug E-4031, which blocks IK in rabbit ventricular myocytes. In single sinoatrial nodal cells, E-4031 (0.1 µmol/L) significantly prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. Half of the cells were arrested completely. At higher concentrations (1 and 10 µmol/L), spontaneous activity ceased in all cells. Three ionic currents fundamental for pacemaking, ie, IK, the long-lasting inward calcium current (ICa,L), and the hyperpolarization-activated current (If), were studied by using the whole-cell and amphotericin–perforated patch technique. E-4031 blocked part of the outward current during depolarizing steps as well as the tail current upon subsequent repolarization (ITD) in a dose-dependent manner. E-4031 (10 µmol/L) depressed ITD (88±4%) (n=6), reduced peak ICa,L at 0 mV (29±15%) (n=4), but did not affect If. Lower concentrations did not affect ICa,L. Additional use of 5 µmol/L nifedipine demonstrated that ITD is carried in part by a calcium-sensitive current. Interestingly, complete blockade of IK and ICa,L unmasked the presence of a background current component with a reversal potential of -32±5.4 mV (n=8) and a conductance of 39.5±5.6 pS/pF, which therefore can contribute both to the initial part of repolarization and to full diastolic depolarization. In conclusion, IK in conjunction with an inward background current plays an essential role in maintaining normal automaticity.
Key Words: sinoatrial nodal cells • class III antiarrhythmic agent E-4031 • delayed rectifier current • impulse generation • background current
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Introduction |
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Diastolic depolarization underlies automaticity of the sinoatrial node (SAN).1 2 3 At least five currents are thought to be involved in diastolic depolarization during normal spontaneous activity in the SAN: (1) an outward potassium current (delayed rectifier, IK),4 5 6 (2) a long-lasting inward calcium current (ICa,L),7 (3) a transient inward calcium current (ICa,T),8 9 (4) a hyperpolarization-activated slow inward current (If),10 and (5) a background current (Ib).11 The decay of IK in conjunction with an inward current is considered to be a major cause of the initiation of diastolic depolarization. If and ICa,T are activated at a relatively negative membrane potential and therefore may contribute to the maintenance of diastolic depolarization. Subsequently ICa,L is activated and promotes the last part of the diastolic depolarization, which gradually turns into the upstroke of the action potential. However, the relative importance of these currents in the process of diastolic depolarization still needs to be determined.
To study the role of IK on impulse generation, we used the class III antiarrhythmic agent E-4031, which is known to block IK in guinea pig12 13 14 15 and rabbit16 ventricles without affecting ICa,L,12 the sodium current,15 or the inward rectifier potassium current.16 Single SAN cells were current- and voltage-clamped to assess the effect of E-4031 on electrical activity and on the three major ionic currents involved in diastolic depolarization, ie, IK, ICa,L, and If.
Our data demonstrate that IK is essential for maintaining normal automaticity by (1) repolarization of the action potential to maximum diastolic potential (MDP) and (2) initiating diastolic depolarization through a decay of the outward potassium conductance together with an inward Ib.
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Materials and Methods |
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Preparation and Solutions
Single cells were isolated from the SAN region by a modified method of DiFrancesco et al.17 Procedures followed were in accordance with institutional guidelines. New Zealand albino rabbits (1.8 to 2.5 kg) of either sex were anesthetized with 1 mL/kg IM Hypnorm (0.3 mg/mL fentanyl citrate and 10 mg/mL fluanisone) under artificial ventilation. The thorax was opened, and 0.1 mL heparin sodium (5000 IU/mL) was injected into the left ventricle. The heart was excised and mounted on a Langendorff perfusion system, and blood was washed out with oxygenated HEPES-buffered solution containing (mmol/L) NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, HEPES 5.0, and glucose 5.5 (normal Tyrode's solution). Temperature was held at 37°C, and pH was adjusted to 7.4 with NaOH. The SAN area was excised and pinned down on a Sylgard layer in a Petri dish containing normal Tyrode's solution. Subsequently, the SAN was cut into four strips (width, ±1 mm; length, ±2 mm) perpendicular to the crista terminalis. These were allowed to equilibrate for 15 minutes in normal Tyrode's solution at room temperature. The strips were placed in a test tube with an oxygenated "calcium-free Tyrode's solution" at room temperature containing (mmol/L) NaCl 140, KCl 5.4, MgCl2 0.5, KH2PO4 1.2, HEPES 5.0, and glucose 5.5. pH was adjusted to 6.9 with NaOH, and the solution was refreshed three times. Next, the strips were incubated at 37°C for 10 to 14 minutes in 5 mL of the "dissociation solution," which was the calcium-free Tyrode's solution to which collagenase B (0.28 U/mL, Boehringer Mannheim), pronase E (0.92 U/mL, Serva), elastase (2.4 U/mg, Serva), and bovine serum albumin had been added. During the incubation, the strips were gently triturated through a pipette with a tip diameter of 2.0 mm. At regular intervals, the solution was microscopically examined for the presence of dissociated myocytes. When single cells appeared, dissociation was immediately stopped, and the strips were transferred into a modified Kraftbrühe (KB) solution18 containing (mmol/L) KCl 85, K2HPO4 30, MgSO4 5.0, glucose 20, pyruvic acid 5.0, creatine 5.0, taurine 30, EGTA 0.5, ß-hydroxybutyric acid 5.0, succinic acid 5.0, Na2ATP 2.0, and polyvinylpyrrolidone 50 g/L (pH was adjusted with KOH to 6.9) and gently shaken. The KB solution was refreshed three times to remove the dissociation solution. Thereafter, the strips were again triturated in KB solution through a pipette (tip diameter, 0.8 to 1.2 mm) for 5 to 10 minutes. Single cells, obtained during this step, were stored at room temperature for at least 45 minutes in KB solution. Samples of the cell suspension (0.4 mL) were placed in a recording chamber on the stage of an inverted microscope (Nikon Diaphot). The cells were allowed to settle for 5 to 10 minutes, after which superfusion (0.6 mL/min) with normal Tyrode's solution was started. For single-cell measurements, we used spindle and elongated spindlelike cells (according to the method of Denyer and Brown19 ), which had not become rounded after readministration of calcium ions to the bathing solution and displayed regular spontaneous electrical activity.
Electrophysiological recordings were performed at a temperature of 35±0.5°C. The temperature of the bathing solution was monitored continuously with a thermistor probe and was maintained by a translucent heating plate underneath the bottom of the recording chamber.20
Electrophysiological Recording
Membrane potentials and currents were recorded by using both
whole-cell21 and amphotericin–perforated patch
techniques.22 23 The whole-cell method was used in only 5
of 10 cells in which the effects of 0.1 µmol/L E-4031 was studied.
The amphotericin–perforated patch technique was used in all other
experiments to reduce dilution of intracellular components, a possible
cause of rundown of membrane currents. Electrodes were pulled from
borosilicate glass (outer diameter, 1 mm; with a glass fiber inside the
lumen) by using a vertical one-stage patch-electrode puller and were
fire-polished. Electrode resistance varied between 3 and 5 M
.
For the whole-cell experiments, electrodes were backfilled with a
micropore-filtered solution containing (mmol/L) potassium gluconate
120, KCl 20, HEPES 5, MgCl2 5, CaCl2 0.6,
Na2ATP 5, cAMP 0.1, and EGTA 5 (pH 7.1). For the
perforated-patch technique, shortly before the experiment we dissolved
6 mg amphotericin B (Sigma Chemical Co) in 100 µL dimethyl sulfoxide,
from which 10 µL was added to a 3-mL electrode solution. Electrode
tips were immersed for 1 s in normal electrode solution and backfilled
with the electrode solution to which amphotericin was added. With this
technique, after sealing to the membrane, a series resistance between 8
and 12 M, which remained stable for at least 1.5 hours, could be
obtained within 10 minutes. This series resistance was compensated for
25% to 6 to 9 M. No attempt has been made to correct for changes
in liquid junction potential. Membrane potential and membrane current
were recorded with a homemade voltage-clamp amplifier. Command
potentials for voltage clamping were obtained from a programmable pulse
generator, which was also used as stimulator. Signals were stored on
videotape (Sony Betamax; bandwidth, 5 kHz) with a pulse code modulation
system (Sony PCM-501), modified to enable DC recordings. Current- and
voltage-clamp recordings were processed off-line by using a custom data
acquisition and analysis program.
Test Protocols and Data Analysis
Action potentials were recorded in current-clamp mode,
off-line–digitized (sample frequency, 2 kHz), and subsequently
analyzed. The following action potential parameters were measured:
action potential amplitude (APA), MDP, diastolic depolarization rate
measured over the first 100 ms starting at the MDP (DDR), maximum
upstroke velocity (dV/dtmax), and duration
between 50% depolarization and MDP (APD100). Cell
capacitance was measured from the initial slope of the transmembrane
voltage in response to current pulses of 50 pA. Mean membrane
capacitance was 52.7±4.9 pF (n=26).
Voltage-clamp recordings were digitized with a sample frequency of 2 kHz for depolarizing voltage-clamp steps; for hyperpolarizing voltage-clamp steps, 500 Hz was used. To discriminate between drug effects and possible rundown of the currents measured, we applied, every 15 s, depolarizing and hyperpolarizing voltage-clamp pulses to the cell starting 2 minutes before and during drug administration. The pulse protocol was as follows: after a conditioning prepulse of 0.5 s to -40 mV, a test pulse to 0 mV of 0.5 s was applied, after which the voltage was clamped back to -40 mV for 0.5 s and then reset in the current-clamp mode. After 15 s, the same pulse protocol was used with a hyperpolarizing test pulse of 1 s to -90 mV. Steady state currents during and tail currents after the test pulse were examined off-line. To study drug effects in more detail, we used depolarizing and hyperpolarizing voltage-clamp steps from a holding potential of -40 mV. Depolarizing test pulses were given with an interval of 1700 ms, and hyperpolarizing test pulses were given at an interval of 3500 ms. Quasi–steady state and instantaneous currents are expressed relative to 0 pA. Tail currents predominantly express deactivation of a current and therefore are expressed relative to the current level at the holding potential. The voltage-clamp protocols are described in more detail in "Results."
For comparison between different cells, whole-cell currents were normalized by dividing by membrane capacitance (pA/pF), unless stated otherwise.
Statistics
For statistical analysis we used the mean values of the
parameters of 10 subsequent action potentials.
All results are presented as mean±SEM. Statistical significance
was determined by application of a Student's t test for
paired observations. A probability P
.05 was considered
significant.
Drugs
E-4031 (1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-methylsulfonyl
aminobenzoyl)piperidine) was a kind gift from Eisai. The agent was
dissolved in distilled water at 1000 times the concentration used.
Nifedipine (Sigma) was dissolved in ethanol (97%, 5 mmol/L). Batches
of both stock solutions were stored at -20°C until use.
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Results |
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Effects of E-4031 on Electrical Activity of Single SAN Cells
We studied the response of the spontaneous activity of single SAN cells to 0.1, 1, and 10 µmol/L E-4031 in the whole-cell and amphotericin–perforated patch voltage-clamp configuration. Fig 1
shows activity of two SAN myocytes before and during
the administration of 0.1 µmol/L E-4031. In Fig 1A the myocyte
remained spontaneously active, as was the case in 5 of 10 cells
studied. In these cells E-4031 caused a prominent depolarization of the
MDP and a large prolongation of the action potential. Furthermore,
cycle length increased, and APA, DDR, and dV/dtmax
decreased. Fig 1B shows the activity from a cell in which spontaneous
activity was completely blocked within 7 minutes after the
administration of E-4031, as was the case in 4 other cells. During
administration of E-4031 (0.1 µmol/L), membrane potential stabilized
at -33±6 mV. Numerical data of action potential parameters are
summarized in the Table. Control action potential
parameters of cells that remained spontaneously active were not
different from those of cells that became quiescent after drug
administration.
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At higher concentrations (1 and 10 µmol/L), we always observed a complete arrest in electrical activity. E-4031 at 1 µmol/L depolarized MDP from -58.8±0.9 to -24.5±1.8 mV (n=3), and E-4031 at 10 µmol/L depolarized MDP from -58.2±2.1 to -19.6±1.8 mV (n=5).
It was questioned whether the various effects on electrical activity could be caused by specific blockade of only one single component of membrane current. Therefore, in a next series of experiments we studied the effects of E-4031 on three ionic currents that are thought to give a major contribution to diastolic depolarization24 : ICa,L, If, and IK.
Effects of E-4031 on Membrane Currents
Specificity of E-4031 as IK Blocker
First, the effect of E-4031 (0.1, 1, and 10 µmol/L) on peak
L-type calcium current was examined. Fig 2A shows
currents elicited during depolarizing voltage-clamp pulses. On
depolarization, a transient inward current, presumably
ICa,L,7 was activated; this response
was not reduced by 1 µmol/L E-4031.
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) of a depolarizing voltage-clamp step from
a holding potential of -40 to 0 mV (test potential
[P1]). The lower tracing shows that the peak
ICa,L was not affected by 1 µmol/L E-4031 (
). Peak
amplitude was measured from the initial peak current transient after
depolarizing steps. B and C, Graphs show current-voltage relations of
ICa,peak obtained by applying voltage-clamp pulses to
various test potentials (P1) from a holding potential of
-40 mV in normal Tyrode's solution and in Tyrode's solution
containing 1 µmol/L E-4031 (B, n=4) and 10 µmol/L E-4031 (C, n=5).
Peak current amplitudes were normalized per cell by dividing them by
the control peak current at 0 mV, averaged, and plotted against
P1.
Fig 2B
summarizes the effect of 1 µmol/L E-4031 on peak current
amplitude at different test potentials (P1). Because of
large differences in peak currents between the cells, we normalized
current amplitudes per cell by dividing them by peak current under
control conditions evoked at 0 mV. These normalized values were
averaged and plotted against P1. Average control peak
current amplitude at 0 mV is -10.6±2.5 pA/pF. The drug (0.1 to 10
µmol/L) did not alter the potential at which the maximum amplitude
was reached. The amplitude of the current was not affected by 0.1 and 1
µmol/L E-4031 (Fig 2B). Fig 2C shows that 10 µmol/L E-4031 reduced
peak current amplitude evoked at 0 mV by 27±13% (n=5), without a
shift in the current-voltage (I-V) relation.
Next, the effect of E-4031 on If was investigated.
Fig 3A shows a current recording in response to a 2-s
hyperpolarizing test pulse (P1) to -90 mV. On
hyperpolarization, an inward current is activated, which at least
partially consists of If,10 and
deactivates after return to the holding potential. In this experiment,
10 µmol/L E-4031 affected neither the quasi–steady state inward
current [If (ss)] nor the tail current of If
(If tail). This is further illustrated in the I-V relations
of If (ss) and If tail (Fig 3B).
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The effect of E-4031 on the total outward current was investigated with
the same protocol used for the measurement of ICa,L. Fig 4A shows a representative current recording in
response to a 500-ms depolarizing test pulse (P1). On
depolarization, ICa,L activates very rapidly, producing a
net inward current. As ICa,L inactivates, the net membrane
current becomes outward, which at least partially is due to the onset
of IK. When the potential returns to holding potential, a
slow decay of the outward current can be observed (tail current), which
was described previously4 16 as being caused by
deactivation of IK. E-4031 (10 µmol/L) reduced both the
quasi–steady state current upon depolarization (ISSD) and
the tail current upon depolarization (ITD).
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The effect of E-4031 (10 µmol/L) on quasi–steady state current
amplitude at different P1 values is summarized in the I-V
relation shown in Fig 4B (n=6). The drug reduced ISSD
amplitude and made the I-V relation more linear. Nevertheless, a
considerable amount of outward current still remained present. Fig 4C shows that E-4031 almost completely reduced tail currents. Tail
current amplitude elicited after a depolarizing voltage step to +20 mV
was reduced from 2.20±0.20 to 0.25±0.08 pA/pF (n=6). Blockade of
steady state outward currents and tail currents was partially
reversible at 0.1 and 1 µmol/L, whereas at 10 µmol/L the drug
effect was irreversible during a washout period of 15 minutes.
Fig 5 summarizes the effect of different concentrations
of E-4031 on ITD, ICa,L, and
If steady state in dose-response curves. ITD
amplitudes were blocked to a large extent (88%). ICa,L was
only significantly reduced at 10 µmol/L E-4031. At lower
concentrations, the drug did not affect ICa,L and
If.
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Even a relatively high concentration of E-4031 does not completely block tail currents after depolarizing voltage-clamp steps. It might be argued that 10 µmol/L E-4031 does not completely block IK. Alternatively, the remaining tail current might be caused by currents other than IK: (1) recovery of inactivation of ICa,L, (2) activation of If, (3) deactivation of the E-4031–insensitive component of IK (ie, IK,s), and (4) a calcium-sensitive outward (potassium) current. A contribution of If is not very likely, considering the small degree and the slow time course of activation at -40 mV.20
Recovery of inactivation of ICa,L or a calcium-sensitive outward current is the most likely cause of the remaining tail currents seen after depolarizing clamp steps. To investigate this possibility, we performed another series of experiments in which the calcium current was blocked by 5 µmol/L nifedipine.
Fig 6A shows current tracings elicited after a
depolarizing test pulse (P1) before (open circles), after
the administration of 5 µmol/L nifedipine (open squares), and after
the administration of 10 µmol/L E-4031+5 µmol/L nifedipine (closed
circles). Nifedipine (5 µmol/L) reduced the peak inward current,
shifted the steady state outward current outward, and reduced the tail
current slightly. E-4031 (10 µmol)+nifedipine (5 µmol/L) blocked
both the fast-activating inward calcium current as well the
time-dependent outward current. After return to the holding potential,
no tail current could be observed. Panels B and C of Fig 6 summarize
the effect of 5 µmol/L nifedipine and 10 µmol/L E-4031+5 µmol/L
nifedipine on quasi–steady state outward current amplitude and tail
current amplitude (n=7). Nifedipine alone shifted the
ISSD-voltage relation slightly outward and reduced the tail
current (11±3%). After the administration of both drugs, the reversal
potential of the steady state I-V is -25 mV, which is close to the
resting membrane potential of -26.7±3.7 mV (n=6). The reversal
potential of the steady state I-V is more negative than the reversal
potential of the steady state I-V after the administration of 10
µmol/L E-4031 alone (-8 mV, Fig 4B). Furthermore, steady state
current amplitudes positive to +40 mV are the same as the control
amplitudes. When both drugs are present, tail current I-V is flat
throughout the whole voltage range (Fig 6C) and does not deviate
significantly from zero.
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) blocks peak inward current, shifts the
quasi–steady state current (ISSD) outward, and reduces
tail current amplitude slightly. E-4031 together with nifedipine
(
These results indicate that the residual tail currents elicited after
depolarizing voltage-clamp steps after the administration of E-4031, as
seen in Fig 4C, are partially caused either by a recovery of
inactivation of ICa,L or by a calcium-sensitive outward
current. After the administration of 10 µmol/L E-4031+5 µmol/L
nifedipine, outward tail currents are lacking in SAN myocytes, thus
excluding the presence of IK,s. These results further show
that 1 µmol/L E-4031 selectively reduces the tail currents elicited
after depolarizing voltage clamp steps (85%+11%=96%).
Discrimination Between IK and Ib
In the previous experiments, it was demonstrated that 10 µmol/L
E-4031 completely but not selectively blocks IK. Complete
blockade of IK enabled us to use E-4031 to dissect
IK from the total membrane current. Nifedipine (5 µmol/L)
was used to exclude calcium current (Fig 6C). To dissect IK
from the total membrane current, we used a protocol that is illustrated
in Fig 7A. From a holding potential of -40 mV, a
conditioning step (P1) to +20 mV was used to activate
IK fully. Thereafter, hyperpolarizing steps to various test
potentials (P2) were made. Current amplitudes after the
step to P2 were measured immediately after the surge of the
capacitive transient. In this way, an instantaneous I-V relation was
obtained, in which IK was fully present.
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). B, Graph
showing the effect of nifedipine and nifedipine with E-4031 on
quasi-instantaneous current (n=7). Current amplitude is measured
relative to 0 pA and expressed as pA/pF. C, Graph showing the
instantaneous current-voltage (I-V) relation of IK obtained
by subtracting the instantaneous I-V obtained after the administration
of nifedipine with E-4031 from the nifedipine instantaneous I-V.
Fig 7A shows representative current tracings of a cell,
recorded under normal conditions (open circles), after the
administration of 5 µmol/L nifedipine (open squares), and in the
presence of 10 µmol/L E-4031+5 µmol/L nifedipine (closed squares).
Nifedipine alone reduces peak inward current during P1 and
also reduces the instantaneous current. The combination of both drugs
blocked time-dependent currents during P1 and
P2 completely. Fig 7B shows the effect of 5 µmol/L
nifedipine and the combination of 5 µmol/L nifedipine and 10
µmol/L E-4031 on the instantaneous I-V relation (n=7). The control
instantaneous I-V curve had a reversal potential of -61.1±1.7 mV.
Nifedipine alone induced a small inward shift of the instantaneous I-V
relation between -60 and 0 mV without a shift in reversal potential.
The application of nifedipine combined with E-4031 resulted in a linear
instantaneous I-V curve with a reversal potential of -32±5.4 mV
and a conductance of 39.5±5.6 pS/pF. We propose that this I-V curve
mainly consists of Ib, which is composed of various
components (see "Discussion"). The difference between the
nifedipine and the nifedipine combined with E-4031 I-V curve can
therefore be considered to be the instantaneous I-V curve of the fully
activated IK (Fig 7C). This is supported by the observation
that this instantaneous I-V difference curve crosses the voltage axis
at -81±3 mV, which is close to the expected reversal
potential25 (see "Discussion").
These experiments demonstrate that the fully activated IK
in SAN myocytes exhibits strong inward rectification (Fig 7C). They
also demonstrate the presence of a substantial Ib with a
reversal potential at -32±5.4 mV (Fig 7B), which renders it
possible that this current contributes to both repolarization and
diastolic depolarization.
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Discussion |
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Effect of IK Blockade on Spontaneous Activity
In the present study, we investigated the role of IK in the impulse generation in rabbit SAN myocytes. The class III antiarrhythmic agent E-4031 was used for blockade of IK.16
Complete blockade of IK with E-4031 could not be obtained
without partial blockade of ICa,L (Fig 5). At lower
concentrations of E-4031 (0.1 and 1 µmol/L), partial but selective
blockade of IK was demonstrated and therefore could be used
to study the role of IK on spontaneous activity. Partial
blockade of IK exerts a complex response on the activity of
isolated SAN cells. Changes in action potential parameters of the nodal
cells are most likely the result of a combination of direct and
indirect effects on various ionic currents. In single SAN myocytes, 0.1
µmol/L E-4031 myocytes increased interval and action potential
duration, depolarized MDP, depressed
dV/dtmax, and reduced DDR. In 5 of 10 cells,
spontaneous activity was completely abolished. Higher concentrations
completely abolished electrical activity. The functional role of
IK and Ib on impulse generation will be
discussed later.
The Delayed Rectifier Current, IK
Even at 10 µmol/L E-4031, tail currents elicited after
depolarizing voltage-clamp pulses were not completely blocked.
Subsequent addition of 5 µmol/L nifedipine caused a disappearance of
tail currents. This suggests that a small part of the tails is caused
by ICa,L or a calcium-dependent outward current. After a
negative voltage-clamp step to a voltage where window calcium current
is present,26 27 recovery from inactivation of
ICa,L will occur. This recovery of inactivation resembles
deactivation of an outward current. An effect of nifedipine on
IK can be excluded, because after the administration of
nifedipine the steady state outward current shifted slightly outward
(Fig 6B). Therefore, complete block of tail currents when both
nifedipine and E-4031 are administered (Fig 6C) is a strong indication
that tails not only represent deactivation of IK
but also, in some part, recovery of inactivation of ICa,L.
Another possibility might be that part of the tail current is due to a
calcium-sensitive outward current, which also will be diminished after
calcium channel blockade. However, the outward shift in the steady
state outward current with nifedipine indicates that such a
calcium-activated potassium conductance is relatively small or even
absent.
Furthermore, these experiments demonstrate that a contribution of
If or a slow component of IK (IK,s)
to the tail currents is absent. The absence of IK,s was
also shown in Fig 6, where after blockade of IK and
ICa,L no time-dependent currents are present, in
agreement with previous single-channel analysis in ventricle
myocytes16 and nodal cells6 of the
rabbit.
Our data show that in a limited concentration range E-4031 is a selective but not complete blocker of IK. It should be emphasized that 1 µmol/L E-4031 selectively blocked IK (96%), when a contribution of a calcium-sensitive component (11%) of the tail current is taken into account. This selective and high percentage of block makes 1 µmol/L E-4031 very suited for use as an IK blocker. Blockade of IK by E-4031 is in agreement with previous reports involving isolated rabbit ventricular16 and guinea pig atrial14 28 myocytes. Our data confirm findings in guinea pig ventricular myocytes where L-type calcium current was not affected by 5 µmol/L E-403112 28 and was only slightly reduced at a concentration of 10 µmol/L.15
Under conditions in which no time dependence in current was seen after
application of E-4031 and nifedipine, we considered IK to
be blocked completely. Thus, we were able to reconstruct the
instantaneous I-V relation of IK as shown in Fig 7C. The
reversal potential of the difference I-V curve (Fig 7C), the fully
activated I-V curve of IK, is -81±3.2 mV (n=7).
Since it was assumed that the ionic mobility of gluconate is
approximately the same as that for lactate, a liquid junction of
-11.4-mV potential was calculated between the pipette solution and
the bath solution.29 Therefore, the actual reversal
potential is -92.4±3.2 mV, which equals the expected reversal
potential (-90 mV).25
The instantaneous I-V curve of IK (Fig 7C) displays strong
inward-going rectification at potentials positive to -30 mV.
Inward-going rectification of IK in the SAN has already
been suggested in experiments by others6 30 31 and is
probably caused by a fast inactivation of IK at positive
potentials.6 16 The rectification is also apparent when
the steady state I-V relations before and after blockade of both
IK and ICa,L (Fig 6B) are compared. At
potentials positive to +40 mV, both I-V relations are superimposable,
which can be explained by assuming that positive to +40 mV, no
IK is present. When we extrapolate the part of the
difference I-V relation with a negative slope (Fig 7C) to the zero
current level, we find a potential at +36 mV, which is close to the
+40 mV that was observed in the steady state I-V relations.
The Background Current, Ib
After the administration of E-4031, a considerable amount of
outward current remained present in the steady state I-V relation
(Figs 4B and 6B) and in the instantaneous I-V curve (Fig 7B). Similar
findings were obtained from guinea pig atrial15 and
ventricular32 myocytes, which were taken as evidence for
two components of IK, one that was blocked
(IK,r) and one that was unaffected (IK,s) by
E-4031. The presence of IK,s can be excluded (see previous
section). We propose that the remaining current after the
administration of E-4031 is not carried through potassium channels but
has a different and possible nonuniform nature and may be considered to
be Ib. The assumption that ISSD is composed of
IK and Ib explains why E-4031 has a much
stronger depressing effect on ITD than on ISSD;
it is because ITD is predominantly IK.
Because time-dependent currents are absent after the administration of
E-4031+nifedipine, the instantaneous I-V relation (Fig 7B, closed
squares) represents Ib also. The background
conductance was 39.5±5.6 pS/pF, with a reversal potential of -32±5.4
mV (n=8), suggestive of a mixed current.
Hagiwara et al11 reported for rabbit SAN the first
evidence of a sodium-dependent Ib with a reversal potential
of -20 mV and and a slope conductance of the I-V relation of 11 pS/pF.
Differences in reversal potential and slope conductance with our
findings can be explained as follows: (1) Besides being composed of
sodium current,11 Ib obtained in our
experiments is likely composed of various other time-independent
currents, such as the Na+-K+
pump,33 Na+-Ca2+
exchanger,34 inward rectifier current,35 and
background Cl- current, which has been described in rabbit
atrium,36 37 although the presence of the latter component
has been questioned in SAN.11 24 38 (2) Hagiwara et
al11 performed their experiments under rather
unphysiological circumstances, by using a rigorous cocktail of blockers
and ion substitutes. In our experiments, we only used two organic
blockers and thus give a more reliable estimate of the total
Ib under physiological conditions.
DiFrancesco39 also reported a total Ib
obtained from the instantaneous current after hyperpolarizing
voltage-clamp steps. The reversal potential was -61 mV (n=5) with a
conductance of 74 pS/pF. A contribution of IK to the
instantaneous current could not convincingly be ruled out, which could
explain the more negative reversal potential and higher slope
conductance.
Comparison of the steady state I-V relation after only IK
blockade (Fig 4B) and the steady state I-V after IK and
ICa,L blockade (Fig 6B) shows that when both currents are
blocked, the I-V curve crosses the zero current level at more negative
potentials. Apparently the quasi–steady state I-V relation after
IK blockade (Fig 4) consists of Ib and also an
ICa,L window current. Blockade of ICa,L by
nifedipine will shift the steady state I-V to more negative potentials
and will make the I-V more linear in the voltage range at which the
ICa,L window current is assumed to be present, between
-50 and +10 mV.8 26 40 The instantaneous I-V relation
after IK and ICa,L blockade (Fig 7B) and the
steady state relation under the same condition (Fig 6B) are quite
similar. Both I-V relations are linear, with an equal slope in the
corresponding voltage range. The steady state I-V relation tended to
cross the zero current level at a somewhat less negative potential
(-25 mV, Fig 6B). This difference in zero current level between
Figs 6B and 7B can only be explained by the presence of an inward
current and is most likely caused by a small fraction of unblocked
ICa,L.
Limitations of the Cell-Attached Patch-Clamp Technique
It can be questioned whether the obtained Ib is caused
only by currents flowing through the cell membrane. We found an
Ib conductance of 2 nS, which corresponds to a
resistance of 0.5 G. This resistance is much smaller than the
apparent seal leakage resistance (Rapp) estimated in our
experiments. Rapp could not reliably be measured because of
the short time between gigaseal formation and the perforating action of
amphotericin, resulting in a rapid reduction in Rapp.
Nevertheless, we could estimate a Rapp of at least 5 G,
which corresponds well to a previously reported value of 5 G (n=25)
measured in nodal myocytes of the rabbit.39
Rapp may even be an underestimate of true seal resistance,
since it includes the parallel combination of seal and patch
resistance.41 Since Rapp is much larger than
the resistance of Ib, it is logical to assume that
the resistance of Ib is predominantly due to intrinsic
membrane properties. When we assume a Rapp of 5 G, this
would result in a corrected reversal potential of Ib of
-35 mV and a conductance of 35.7 pS/pF, which are both very close to
the uncorrected values.
Functional Role of IK and Ib in
Spontaneous Activity
When the findings involving IK and Ib are
combined, the following description of their functional role in
pacemaking can be made: We have shown that the inward going
rectification of IK is so strong that at potentials
positive to +10 mV, Ib is even larger than IK.
Therefore, Ib is a dominant source of repolarization in the
first part of the repolarization process and provides an explanation
for the relatively small effects of E-4031 on the early part of the
repolarization, when a large fraction of IK is blocked (Fig 1A). During the second part of repolarization, IK recovers
from inactivation and carries sufficient current to repolarize the
membrane to MDP. During diastole, IK deactivates, and in
conjunction with an inward current, the membrane starts to depolarize.
During diastole, Ib is inward and will therefore contribute
to whole diastolic depolarization as well. Contributions of
ICa,L and If to diastolic depolarization are
also likely to occur.24
|
Acknowledgments |
|---|
This work was supported by a grant from the Netherlands Organization for Scientific Research and the Netherlands Heart Foundation (NWO grant 900-516-093). The authors thank Tobias Opthof, PhD, Department of Experimental and Clinical Cardiology, University of Amsterdam, and Ronald Wilders, PhD, Department of Physiology, University of Amsterdam, for their stimulating interest and helpful discussions; Kohei Sawada, MD, PhD, Tsukuba Research Laboratories, Eisai, for providing E-4031 and helpful suggestions; and Jan Zegers, MSc, Department of Physiology, University of Amsterdam, for his excellent technical support in constructing an action potential analysis program.
Received April 14, 1994; accepted December 6, 1994.
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References |
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