Extracellular K1 Dependence of Inward Rectification
Kinetics in Human Left Ventricular Cardiomyocytes
Patrick Bailly, PhD; Maud Mouchonière, PhD; Jean-Pierre Bénitah, PhD; Lionel Camilleri, MD;
Guy Vassort, ScD; Paco Lorente, MD
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Background—In human ventricular cells, the inwardly rectifying K1 current (IK1) is very similar to that of other
mammalian species, but detailed knowledge about the K1-dependent distribution of open and blocked states during
rectification and about the K1-dependent modulation of inactivation on hyperpolarization is currently lacking.
Methods and Results—We used the whole-cell patch-clamp technique to record IK1 in myocytes isolated from
subendocardial layers of left ventricular septum from patients with nonfailing hearts with aortic stenosis and cardiac
hypertrophy who were undergoing open-heart surgery. Outward currents were very small at voltages positive to the
reversal potential but increased at high external [K1]. Chord conductance measurements and kinetic analyses allowed
us to estimate the proportion of channels in the open state and of those showing either slow unblock or instantaneous
unblock (the so-called slow or instantaneous “activation”) on hyperpolarization: the distribution in the individual states
was dependent on external [K1]. The proportion of channels unblocking slowly was greater than that of channels
unblocking instantaneously on hyperpolarization from the plateau voltage range. Hence, because of the previously
reported link between the presence of highly protonated blocking molecules and slow unblock kinetics, it is suggested
that high cellular concentrations of spermine may account for the low outward current density recorded in these cells.
The current decrease observed on extended hyperpolarization was significantly relieved by an increase in external [K1].
Conclusions—The pattern of IK1 current alterations observed in the present model of human ventricular hypertrophy might
favor enhanced excitability and underlie ventricular arrhythmias, possibly via increased intracellular polyamine levels.
(Circulation. 1998;98:2753-2759.)
Key Words: potassium n myocytes n electrophysiology
he inward rectifier K1 current (IK1) is a major determinant of a stable resting potential in excitable cells.1
Mediating a reduced flow of outward currents at voltages
positive to reversal potential, inward-going rectification also
allows development of the long plateau phase of the cardiac
action potential2 and sets the excitability threshold for depolarizing currents.3 Furthermore, it has been suggested that
outward currents through IK1 channels contribute to the final
phase of repolarization of the action potential.4 – 6
The steep inward rectification of IK1 around the reversal
potential (Erev) is typically associated with a time- and voltagedependent current increase after an instantaneous current jump
on hyperpolarization. In the 1980s, it was shown that a voltagedependent block by internal Mg21 ions contributes to strong
rectification,7 mainly at depolarized levels far from Erev.8 However, the fast kinetics of the Mg21 block cannot account for the
observed activation kinetics, because in the absence of Mg21,
inward rectifier K1 channels still rectify strongly, and the slow
open-close transitions of the so-called activation gate remain
little affected.8 Indeed, recent studies on channels exogenously
expressed from cloned inward rectifier K1 channel genes,
Kir2.19 and Kir2.3,10 have shown that the intrinsic gating reflects
the blocking and unblocking kinetics of intracellular protonated
polyamines.11–18 Regulatory mechanisms are reportedly involved
in these processes: on the one hand, the higher the cationic
charge of the polyamine, the slower the time course of recovery
from the polyamine-induced block14,19; on the other hand, cloned
channels, like native IK1 channels, exhibit clear relief of rectification after external K1 concentration ([K1]o) increases.20
In humans, a few recent data suggest that the characteristics of
ventricular native IK1 channels are similar to those of other
mammalian hearts,21–24 except for smaller outward current amplitudes than in other species.2,3,5,25 No information is currently
available on the [K1]o dependence of unblocking and blocking
kinetics or on the conductance properties of the native human
ventricular IK1. In the present article, we examine the influence of
[K1]o changes on the voltage dependence of nonconductive
states induced by depolarizing pulses and on the “inactivation”
kinetics observed during prolonged hyperpolarization in human
ventricular cells from patients without left ventricular (LV)
T
Received March 16, 1998; revision received July 29, 1998; accepted August 13, 1998.
From the Département de Chirurgie Cardiovasculaire, Hôpital Gabriel Montpied, Clermont-Ferrand (P.B., L.C.), and U 390 INSERM, CHU Arnaud
de Villeneuve, Montpellier (G.V., P.L.), France. Dr Bénitah is now a postdoctoral fellow in the Division of Cardiology, Johns Hopkins University School
of Medicine, Baltimore, Md. Dr Mouchonière is now a postdoctoral fellow in the Institut National de Recherche Agronomique, Theix, France.
Correspondence to Dr Paco Lorente, U 390 INSERM, CHU Arnaud de Villeneuve, 34295 Montpellier, France. E-mail [email protected]
© 1998 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
2753
2754
IK1 Current in the Nonfailing Human Heart
dysfunction. We report that the [K1]o dependence in the distribution of blocked states on depolarization in isolated human
ventricular cells follows a pattern similar to that observed in
Kir2.1 exogenously expressed channels.6 The hyperpolarizationinduced “inactivation” may be accounted for both by extracellular Na1 block and by external K1 depletion, possibly facilitating a polyamine-induced channel reblock; this process seems to
be significantly relieved by an increase in [K1]o.
Methods
Characterization of Patients
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Ventricular tissue samples were obtained from 21 patients undergoing corrective cardiac surgery for acquired aortic stenosis with LV
hypertrophy but without LV dysfunction. Patient age was 63.568.6
years (range, 48 to 78 years); 12 were men, 9 women. Clear evidence
of LV hypertrophy was based on echocardiographic and ECG
findings (echocardiographic septal thickness was 13.263.0 mm;
normal values, ,10 mm). All the patients had compensated hypertrophy (NYHA functional class II; cardiac index, 2.6760.51 L z
min21 z m22; ejection fraction, 0.6360.15; LV end-diastolic pressure,
14.063.1 mm Hg; echocardiographic shortening fraction,
0.3560.06) and hence were not receiving any cardiotonic drugs. All
patients gave written informed consent to the study protocol, which
had previously been approved by the institutional committee on
human investigation and complies with principles outlined in the
Declaration of Helsinki.
Cell Isolation
Endocardial LV septal sampling was performed through the aortic
orifice during circulatory arrest. Specimens were taken from the
muscular septum just beneath the commissure between left and right
coronary leaflets of the aortic valve. They consisted of scalpel
shavings from the LV septal wall ('10 to 12 mm long, 5 to 7 mm
wide, ,3 mm thick, 0.1 to 0.6 cm3), in every case superficial
(,3-mm depth). Immediately after the specimens were taken, they
were placed in cardioplegia solution bubbled with 100% O2 at 19°C.
Experimental procedures began within 15 minutes of removal.
Ventricular myocytes were isolated by an enzymatic dissociation
method as described previously.26 Briefly, chunks were incubated at
35°C for 30 minutes in a Ca21-free Tyrode’s solution supplemented with
300 IU/mL collagenase V, 4 IU/mL protease XXIV, and 1 mg/mL BSA
(Sigma). Then, the supernatant was removed and replaced by a fresh
enzyme medium having the same composition but without protease.
When the yield appeared to be maximal, minced tissue was strained
through a 200-mm nylon mesh to remove debris and undigested tissue.
Cells were then suspended in Ca21-free Tyrode’s solution and stored for
1 hour at room temperature (20°C to 22°C) before the experiment was
begun. Only quiescent rod-shaped cells showing clear striations without
significant granulation were used.
Recording Techniques
Current recordings were obtained by the standard whole-cell recording
technique with an Axopatch 1D amplifier (Axon Instruments) with a
100-MV feedback resistance headstage, the 125-kHz Labmaster board,
and pClamp program V 5.5.1 (Axon Instruments). Microelectrodes
pulled from soft glass capillary tubing (1.5 to 1.6 mm OD) had tip
resistances ranging from 1 to 1.5 MV. A silver–silver chloride pellet
encased in a 3 mol/L KCl agar bridge was placed in the bath and used
as the ground reference electrode. Cell capacitance and series resistance
were measured by 610-mV voltage steps applied from a 270-mV
holding potential and calculated as previously described.26 Series resistance was kept ,5 MV (2.2661.04 MV; n542) and was compensated
by 60% to 80%; the time constant of the capacitive current decay was
0.1960.04 ms after compensation. The electrode potential was adjusted
to zero after immersion of the pipette tip; this zeroing caused a positive
voltage bias that was not corrected. Currents were low-pass filtered at 2
kHz, digitized at a sampling interval of 200 ms, and stored for off-line
analysis.
Solutions
The transport solution contained (in mmol/L) NaCl 147, KCl 20,
CaCl2 2, MgCl2 16, glucose 6, and HEPES 5 (pH adjusted to 6.8 with
KOH). For cell isolation and cell storage, the Ca21-free Tyrode’s
solution had the following composition (in mmol/L): NaCl 120, KCl
4, MgCl2 1, HEPES 10, and glucose 6; pH was adjusted to 7.4 with
NaOH. For study of IK1 kinetics and gating properties, the standard
Tyrode’s solution contained (in mmol/L) NaCl 130, KCl 4, CaCl2 2,
MgCl2 1.1, mannitol 0.4, HEPES 25, and glucose 11; pH was
adjusted to 7.4 with NaOH. In experiments designed to study the
[K1]o dependence of inward rectification kinetics, different [K1]o
were set by equimolar substitution of KCl for NaCl in the Tyrode’s
solution. Patch pipettes were filled with an internal solution containing (in mmol/L): KCl 120, MgCl2 1, Mg-ATP 3, Tris-GTP 0.4,
EGTA 10, HEPES 25, and glucose 10 (pH adjusted to 7.2 with
KOH). The addition of EGTA buffer to the internal solution aimed
at minimizing calcium-activated outward currents and the absence of
Na1 ions in the internal solution were also expected to inhibit
calcium influx through Na1-Ca21 exchange. Ca21 current was inhibited by 2 mmol/L Co21, Ito was inhibited by 3 mmol/L 4-aminopyridine, and Na1 current was minimized by the voltage-clamp protocol.
Kinetic Analysis
The results are expressed as mean6SEM. As recently reported by
Ishihara,6 current kinetics relative to channel states were analyzed at
the end of depolarizing pulses, as follows. The proportion of the
channels residing in the open state (PO) was estimated from the chord
conductance, G, calculated as the current level: driving force ratio
G5I/(Vm2Erev), where Vm is membrane potential. Then, G was
normalized to its maximum value, Gmax, obtained at Vm52130 mV,
which was '50 mV negative to Erev: PO5G/Gmax. Single Boltzmann
fits were used to describe the voltage dependence of PO.
It was clear from tail currents at 2130 mV after depolarizing steps
that channels were in at least 2 nonconductive states at the end of the
preceding potential: one that opened instantaneously on hyperpolarization (the proportion of channels in this state was called PC,Inst) and
another that activated in a time-dependent manner (the proportion of
channels in this state was called PC,Slow). The analysis of inward
currents recorded by hyperpolarizing the membrane from various
levels to 2130 mV allowed us to obtain PC,Inst and PC,Slow. The
time-dependent increase of inward currents was best fitted by a
single exponential function of the form A exp[2(t2k)/t]1C, and the
current level at the onset of voltage change, I(0), was obtained by
extrapolating back the fitted curve to the beginning of the test pulse.
The amplitude of the time-dependent component relative to the
maximum level C of the inward current at 2130 mV, [C2I(0)]/C,
was assumed to give an estimate of PC,Slow at the membrane potential
that preceded hyperpolarization. On the basis of previously reported
works,6,14,19 we hypothesized that PC,Slow represented the proportion of
channels blocked by the most positively charged polyamines just
before hyperpolarization.
An estimation of the probability of the channel being in the
nonconductive state that opens instantaneously on hyperpolarization
was provided by PC,Inst5[I(0)/C]2PO, ie, the fraction of instantaneous
outward currents not attributable to channels open at the end of
previous conditioning potentials. We hypothesized that PC,Inst represented the proportion of channels blocked by the least positively
charged polyamines and by Mg21 ions just before hyperpolarization.
In contrast, the current decline during prolonged hyperpolarizations was best fitted by a double exponential function of the form Af
exp[2(t2k)/tf]1As exp[2(t2k)/ts]1B, where tf and Af are the time
constant and initial amplitude of the fast component, ts and As the
time constant and initial amplitude of the slow component, and B the
steady-state component.
Subsequently, “activation” and “inactivation” are written in quotes
because they describe increase and decrease in current during voltage
steps but do not refer to their classic meaning.
Results
The cell membrane was held at 250 mV (Vh), '40 mV
positive to the predicted K1 equilibrium potential (EK).
Bailly et al
December 15, 1998
2755
Increasing [K1]o induced a depolarizing shift in Erev, an
increase in the peak current and slope conductance, and a
slight increase of outward currents in 20 mmol/L [K1]o
(Figure 2A). Linear fitting to mean Erev plotted against log
[K1]o yielded a slope of 51.563.0 mV, close to the 58.5 mV
expected from the Nernst equation (Figure 2B), thus suggesting that the main charge carriers for the background current in
human ventricular cells are K1 ions. The square-root dependence of conductance of inward rectifiers on [K1]o2,25,27 was
also a property of the human ventricular macroscopic IK1
(Figure 2C): slope conductances measured from the linear
portion of the I-V relationship at potentials negative to Erev
were plotted versus [K1]o and fitted by a linear regression
function. The mean slope of regression lines obtained from
individual cells was 0.5460.19 (n511).
“Activation” (Unblock) Kinetics
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Figure 1. Whole-cell IK1 in human ventricular myocytes. A, Current records in a representative cell (Cm574 pF). B, Mean peak
(M) and steady-state (F) current densities obtained in 16 cells
bathed in 4 mmol/L [K1]o. In all figures, SEMs are shown when
they are larger than symbols.
Currents were elicited by 200-ms voltage-clamp steps applied
from Vh to 2170 to 220 mV at 0.16 Hz. After a 5-ms return
to Vh, a 50-ms voltage-clamp pulse was delivered to 2140
mV. Inward rectification was evidenced by larger current
amplitudes at voltage steps negative to 280 mV (Figure 1A).
The “activation” phase of the inward current speeded up as
voltage steps were made more negative. A significant decay
of inward current (“inactivation”) occurred at voltages negative to 2100 mV, and marked declines at stronger hyperpolarizations caused a negative slope region in the steady-state
relation (Figure 1B). Erev was between 280 and 270 mV, and
for less negative potentials, very small outward currents gave
rise to a flat zone without any negative slope conductance; on
the average, the maximum outward current density was
0.5760.15 pA/pF at 250 mV.
As in native and cloned channels,20,25 the “activation” phase
of current followed a monoexponential, voltage-dependent,
and K1-sensitive time course (Figure 3). The time constant
decreased as the test potential was made more negative,
whereas increasing [K1]o caused a rightward shift of the
“activation” time constant, tact, without any appreciable
change in the voltage sensitivity. Time constants decreased
e-fold for a 32.5-, 34.1-, and 33.1-mV hyperpolarization at 4,
8, and 20 mmol/L [K1]o, respectively. When the time constants at different [K1]o values were replotted against Vm2EK
to account for the shift in Vrev induced by increasing [K1]o, the
data points were superimposed along the same line (not
shown).
It is currently acknowledged that “activation” of inward
currents results from the relief of Mg21 and polyamine
channel block from the channels11,12,14 and can follow a
multiphase time course.6,14 Unblock “activation” kinetics
were described by analyzing tail currents on hyperpolarization to 2130 mV after various voltage steps (Figure 4A). The
individual fits superimposed on current traces show that the
exponential increase in inward currents started from 91%,
Figure 2. Effects of [K1]o changes. A, a,
Mean peak-voltage relationships from 7
cells successively exposed to 4 (F), 8
(M), and 20 (Œ) mmol/L [K1]o. A, b, Same
data between 260 and 220 mV with
expanded ordinates. B, Dependence of
Erev on [K1]o. Semilogarithmic plot of Erev
determined at 4, 8, and 20 mmol/L [K1]o
(n511). Solid line indicates linear fit to
data points; dashed line, Nernst equation. C, Bilogarithmic plot of slope conductance vs [K1]o.
2756
IK1 Current in the Nonfailing Human Heart
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Figure 3. Unblock activation kinetics. A, Inward currents elicited
by 200-ms pulses delivered from 250 mV (Vh) to 2140 to 2170
mV in a cell bathed in 4 mmol/L [K1]o. Monoexponential fits with
t values noted near each evoked current. Cm5131 pF. B, [K1]o
and voltage dependence of activation time constants (tact) in 4
cells successively exposed to 4 (F), 8 (M), and 20 (Œ) mmol/L
[K1]o. Fitting equation: t5K exp(Vm/s), where Vm is membrane
potential and s slope factor.
64%, and 36% of the maximum inward current on hyperpolarization from 2100, 290, and 250 mV, respectively.
Because the relative amplitude of the time-dependent component, PC,Slow, is assumed to reflect the proportion of channels blocked by the most protonated polyamines at the end of
prepulses,6 these findings suggest that this proportion in-
Figure 4. Distribution of channel states: PO, open; PC,Slow,
blocked by most protonated polyamines; PC,Inst, blocked by least
protonated blocking molecules: evaluated at end of 20-ms
pulses applied as shown in inset. A, Currents on hyperpolarization from 2100 (E), 290 (), and 250 (f) to 2130 mV, with
superimposed fits (tau ranging between 1.7 and 2.0 ms for all
traces). B, PO curves at 4 (F), 8 (M), and 20 (Œ) mmol/L [K1]o
(n56). C, Voltage dependence of PC,Slow and PC,Inst in 4 mmol/L
[K1]o (n56). D, Voltage dependence of PC,Slow and PC,Inst in
8 mmol/L [K1]o (n55).
creased when prepulse levels were successively depolarized
from 2100 to 250 mV. Conversely, the proportion of
channels blocked by the least protonated molecules, PC,Inst,
corresponds to the value 12PC,Slow2PO, where PO is the
probability of the channel being opened in the preceding
membrane potential. From the I-V relations obtained at the
end of 20-ms prepulses, the voltage dependence of PO was
estimated at different [K1]o successively applied (Figure 4B).
Increasing [K1]o augmented PO (ie, decreased channel block)
at any given membrane potential and caused an obvious PO
shift to more positive potentials. To describe the [K1]o
dependence of inward rectification, the membrane potential at
which channels were half-blocked (V1/2) was obtained from
single Boltzmann fits to experimental PO values, and its
dependence on EK was examined. In 4, 8, and 20 mmol/L
[K1]o, V1/2 and EK mean values were (in mV) 292.3, 290.4;
274.2, 272.8; and 248.5, 249.5, respectively. Hence,
inward rectification shifted proportionally to the change in
EK, and a linear approximation to these data gave a slope
factor of 1.07. PC,Slow and PC,Inst at the end of prepulses were
estimated from the kinetic analysis of currents in the following hyperpolarization (Figure 4C and 4D). Steep increases in
PC,Slow were observed at potentials around EK (290 and 273
mV at 4 and 8 mmol/L [K1]o, respectively), but PC,Slow
decreased only to a moderate extent at more depolarized
potentials. PC,Inst increased in a voltage-dependent manner at
both [K1]o but did not cross over the PC,Slow-voltage relationship within the voltage range studied, in contrast to previously reported findings.6
“Inactivation” Kinetics on Hyperpolarization
The time-dependent decline of IK1 on hyperpolarization has been
referred to as “inactivation.”25,28,29 As previously reported,28 the
best fit to current traces was also provided in our cells by 2
exponentials demonstrating a clear dependence on voltage. On
average, at 2120, 2130, 2150, and 2170 mV, the time
constants of the fast and slow components, tf and ts, were (in
ms) 30, 243; 25, 135; 16, 53; and 11, 33, respectively.
The [K1]o dependence of “inactivation” kinetics was investigated in cells successively bathed in 4, 8, and 20 mmol/L
[K1]o and using voltage steps to 2170 mV (Figure 5A). As
[K1]o was raised, both peak (Ipeak) and steady-state (Iss)
currents were increased and the time course of “activation”
was accelerated. However, “inactivation” was slowed down,
resulting in a decrease of the relative amount of current
“inactivated” at the end of the pulse. If we define the
difference between the peak and steady-state current as Iinact,
then the Iinact/Ipeak ratio was 0.80, 0.63, and 0.23 at 4, 8, and
20 mmol/L [K1]o, respectively. This behavior was observed
in 5 of 5 cells studied.
It was also of interest to examine the classic steady-state
inactivation relationship (Figure 5B). Normalized currents
plotted versus prepulse potentials were well fitted with the
function I/Imax51/{11exp[(V1/22Vm)/k]}1B, where B is the
steady-state baseline level. In 4 and 8 mmol/L [K1]o, V1/2 (Vm
for I/Imax50.5) and k were (in mV) 2154.862.1, 8.161.1
mV and 2161.961.3, 11.061.3, respectively; in 20 mmol/L
[K1]o, k was 22.262.8 mV. For potentials more negative than
2140 mV, the availability was higher with increasing [K1]o:
Bailly et al
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the calculated steady-state baseline level was 0.1960.02,
0.3160.03, and 0.6060.05 in 4, 8, and 20 mmol/L [K1]o,
respectively.
The time-dependent decrease of IK1 during hyperpolarizing
pulses was attributed in part to extracellular K1 depletion in
isolated feline ventricular myocytes.25 The notion that depletion may also occur in human ventricular myocytes was
tested with a 2–pulse tail protocol in 4 mmol/L [K1]o (Figure
5C). Erev deduced from tail-current measurements was compared with that obtained from the steady-state I-V relationship. At the end of the conditioning pulse, there was a
28.2-mV shift of Erev relative to the one determined from the
steady-state I-V relationship. On average, the negative shift
of Erev related to extracellular K1 depletion amounted
27.061.3 mV (n57). Assuming that intracellular K1 activity
does not change, this shift in Erev corresponded to a decrease
of 1.1 mmol/L in [K1]o (Figure 2B). From the expected
change in conductance resulting from this [K1]o decrease
(Figure 3C) and the consequent change in driving force, we
may expect a current decline of '24% at 2170 mV caused
by depletion of extracellular K1 in the unstirred solution layer
close to the cellular surface. Because the difference between
peak and steady state at 2170 mV shown in Figure 1B
suggests a mean decrease of 73% in IK1 conductance, depletion might account for '33% of the reduction in IK1 conductance at 2170 mV.
Discussion
The major findings of the present study are threefold. (1) The
nonconductive states in human ventricular IK1 channels follow a
[K1]o dependence analogous to that observed in expressed
Kir2.1 channels.6 (2) The distribution pattern of blocked states
suggests a more prominent binding to high-affinity than to
low-affinity blocking molecules within the plateau voltage
range. (3) There is clear evidence that increasing [K1]o may
relieve the “inactivation” induced by strong hyperpolarizations.
[K1]o Dependence of IK1 Conductance in Human
Ventricular Myocytes
The square-root dependence of conductance on [K1]o is a
property of native and cloned inward rectifier channels from
mammalian tissues.2,9,10,25 Exogenous expressions of cloned
channels have demonstrated that inward currents are substantially inhibited by polyamines over the full range of voltages
negative to EK,11,16 raising the question of possible alterations
of inward conductance in native channels as well. Therefore,
the conductance-[K1]o relationship may depend on several
polyamine-related factors.20 Indeed, an approximate squareroot dependence can be found in macroscopic native currents
(eg, in the present study), possibly because conductance
measurements are performed at [K1]o much lower than the
expected internal K1 concentration. In this case, it is recognized that IK1 channels behave as unsaturated open-channel
pores at potentials negative to EK, and the square-root
dependence of conductance on [K1]o just reflects this
behavior.27
1
[K ]o Dependence of Unblocking Kinetics
We previously showed that action potential duration (APD) is
prolonged in human LV septal cells from nonfailing hyper-
December 15, 1998
2757
trophied hearts, and we attributed this pattern partially to a
dramatic downregulation of Ito.30 However, outward IK1 currents also contribute to the final phase of repolarization.4 – 6
Accordingly, the rate of late repolarization, which was found
to be slower in hypertrophied cells than in control cells,
prompted us to search for alterations in IK1 current. We found
only very weak outward currents at potentials positive to Erev
compared with those reported in other mammalian species.2,3,5,25 This was in contrast to the observations of Koumi et
al24 but consistent with other studies on human ventricular
cells21–23 and may suggest the presence of high cytoplasmic
polyamine levels17 and particular distributions of IK1 channel
states on depolarization.6
The assumed distribution of channels in the individual
states was apparently dependent on [K1]o (Figure 4). Since
the PO fitted curve reflected the voltage dependence of inward
rectification, the [K1]o-dependent shift of inward rectification
was estimated by the V1/2'EK relationship, which demonstrated high sensitivity on [K1]o, because the slope between
V1/2 and EK was 1.07, a value close to that observed for
spermine in inside-out patches from Xenopus oocytes expressing Kir2.1.20 The voltage and [K1]o dependence of PC,Inst
and PC,Slow was reminiscent of that established for the proportion of channels blocked by Mg21 (PMg) and spermine (PSpm) in
murine fibroblast cells,6 with a positive shift of '20 mV by
increasing [K1]o from 4 to 8 mmol/L. However, in contrast to
the latter reported findings, no relationship crossover was
found; the fraction of channels blocked by highly protonated
molecules remained greater than the fraction of channels
blocked by weakly protonated molecules within the plateau
voltage range. This pattern must be interpreted by considering
the reported ability of Mg21 and putrescine (Put21) to increase
outward currents as follows.6 Given that Put21 and Mg21
blocking rate constants are larger than that of spermine,
Put21/Mg21-blocked states can reach higher proportions than
spermine-blocked states (PSpm) at short depolarizing voltage
steps. Thereafter, the highest affinity of spermine with the
channel induces time-dependent redistribution of Put21/Mg21blocked to spermine-blocked states, and this occurs through
an intermediate open state. As a result, the larger the number
of Mg21/Put21-blocked channels are, the more channels reside
in the open state before passing into the spermine-blocked
state, thus increasing outward current amplitude. However,
because blocking rates depend on the concentrations of
blocking molecules, an increase in spermine concentration
may entail negative and upper shifts of PSpm at the expense of
PMg/Put and thus reduce outward currents because of the
competitive binding of blocking molecules to the channel.6
Therefore, the higher values of PC,Slow within the plateau
voltage range compared with PC,Inst might be indicative of
relatively high levels of spermine concentration, underlying
decreased availability of outward current and hence enhanced
excitability.3
[K1]o Dependence of Current “Inactivation”
Increasing [K1]o apparently relieves IK1 “inactivation” during
hyperpolarizing pulses (Figure 5A). The steady-state “inactivation” shown in Figure 5B also favors the notion that high
[K1]o significantly alleviates “inactivation,” because after
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IK1 Current in the Nonfailing Human Heart
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Figure 5. [K1]o-induced relief of “inactivation.” A through C,
Cells are successively exposed to 4 (F), 8 (M), and 20
(Œ) mmol/L [K1]o. A, Currents elicited in 1 representative cell
(Cm580 pF) by a 2170-mV pulse followed by a pulse to 2140
mV (Vh5250 mV). Bottom 2 dashed lines show amplitude of
current “inactivated” (Iinact) during test pulse at 20 mmol/L [K1]o,
calculated as difference between peak (Ipeak) and steady-state
(Iss) currents. For clarity, currents “inactivated” at 4 and
8 mmol/L [K1]o are not represented. B, Steady-state “inactivation” of inward current studied with 2-pulse protocol shown in
inset. Currents measured 2 ms after onset of test pulse (TP)
were normalized to maximum value obtained with conditioning
pulses (CP) that totally removed “inactivation” and were plotted
against CPs. C, Steady-state current-voltage relation established with protocol shown in Figure 1 (E), together with steadystate (f) and instantaneous (M) tail currents obtained with a
2-pulse tail protocol. Inset, Currents elicited in a cell during latter protocol applied to determine EK: from a 250-mV Vh, prepulse was to 2170 mV for 80 ms, and subsequent test pulses
were applied to 2120 to 210 mV in 5-mV increments. Tail currents were extrapolated by fitting to beginning of test pulses.
Cm5112 pF. Steady-state currents reversed at 8.2 mV less negative than tail currents.
strong hyperpolarizing pulses, the current availability is
'3-fold greater in 20 than in 4 mmol/L [K1]o.
The time-dependent decline of inward IK1 on hyperpolarization in physiological solution was classically ascribed to a
voltage-dependent block of the channel by external Na1,
reportedly found in native and cloned inward rectifiers.9,28,29
This view is supported by the disappearance of the negative
slope in the steady-state current-voltage relationship when
Na1 substitutes are used.25,28,29 However, this phenomenon
may develop in the absence of external Na1 in guinea-pig
ventricular cells31 and in Kir2.3 channels expressed in Xenopus oocytes.14 In contrast, the notion of [Na1]o block–induced
“inactivation” has emerged from the use of various Na1
substitutes subsequently considered inhibitory28 or even as
open-channel blockers.32 Indeed, it is unlikely that [Na1]o acts
as an open-channel blocker, but it can redistribute channels to
a closed state, thus contributing to the decline of current
during prolonged hyperpolarization.32 This may be reconciled
with the multiple-ion-block hypothesis that suggests interactions between permeant and blocking ions at multiple binding
sites.1 Accordingly, Na1/K1 competition in the channel pore
may be an important mechanism by which increasing [K1]o
can relieve “inactivation.”
Extracellular K1 depletion may also account for some
decrease in IK1 conductance during hyperpolarization25 and is
presumably amplified by the structure of the T-tubular
system. The K1 selectivity filter, cradled at the external
mouth of the pore,33 and the deep, high-affinity binding sites
for polyamines probably underlie the key structures governing the selective K1 conduction in IK1 channels through
electrostatic interactions.20 Thus, the extracellular K1 depletion induced by hyperpolarization might reduce K1 occupancy at the selectivity filter and thus decrease interactions
with polyamine binding sites; this should allow partial reblock of the channel by polyamines and might contribute to
the “inactivation” process. Because relative changes of extracellular K1 in restricted spaces are expected to be less
important in high than in low [K1]o, this “interactive”
mechanism might also contribute to the relief of “inactivation” induced by high [K1]o together with the Na1/K1
competition mentioned above.
Physiological Implications
Extended depolarization decreases outward current by
strengthening high-affinity spermine block.6,19 A depolarizing
shift of Vh also reduces it by increasing PSpm and diminishing
the availability of channels to be blocked by Mg21 on
depolarization.6 Therefore, APD prolongation usually observed in the present model30 or slight resting potential
depolarizations can reduce the proportion of Mg21/Put21blocked channels during the plateau, then decreasing the
outward current flow during repolarization and further
lengthening APD. Conversely, recent reports15,17 have demonstrated that manipulation of polyamine levels can bring
about dramatic changes in the repolarization phase and
significantly increase excitability. Hence, IK1 alterations during evolving cardiac hypertrophy in patients with aortic
stenosis may favor enhanced excitability and arrhythmias,
insofar as elevated polyamine levels have been found in
hypertrophy models.17
In other aspects, the question remains whether the depolarizing shift of the voltage dependence of blocked states
induced by [K1]o increases might contribute to the APD
lengthening observed at the initial stage of acute myocardial
ischemia.34 Indeed, the possibility cannot be ruled out that the
cellular K1 loss subsequent to acidification at the early stage
of acute ischemia may entail a rightward shift of blockedstate relationships. A subsequent increased prevalence of
high-affinity polyamine-blocked states in the plateau voltage
range might be expected from the observed patterns (Figure
Bailly et al
4), with a resulting decrease in outward current availability
and AP prolongation.
Acknowledgments
This work was supported in part by European grant PL 950287,
BIOMED II. We gratefully acknowledge Dr Yves Tourneur for
reading the manuscript and J.P. Da Ponte for his valuable
technical assistance.
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Extracellular K+ Dependence of Inward Rectification Kinetics in Human Left Ventricular
Cardiomyocytes
Patrick Bailly, Maud Mouchonière, Jean-Pierre Bénitah, Lionel Camilleri, Guy Vassort and Paco
Lorente
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Circulation. 1998;98:2753-2759
doi: 10.1161/01.CIR.98.24.2753
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