(Circulation. 1998;98:2753-2759.)
© 1998 American Heart Association, Inc.
Basic Science Reports |
Extracellular K+ Dependence of Inward Rectification Kinetics in Human Left Ventricular Cardiomyocytes
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 paco{at}u390.montp.inserm.fr
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Abstract |
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Background—In human ventricular cells, the inwardly rectifying K+ current (IK1) is very similar to that of other mammalian species, but detailed knowledge about the K+-dependent distribution of open and blocked states during rectification and about the K+-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 [K+]. 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 [K+]. 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 [K+].
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.
Key Words: potassium • myocytes • electrophysiology
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Introduction |
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The inward rectifier K+ 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 5 6
The steep inward rectification of IK1 around the reversal potential (Erev) is typically associated with a time- and voltage-dependent current increase after an instantaneous current jump on hyperpolarization. In the 1980s, it was shown that a voltage-dependent block by internal Mg2+ ions contributes to strong rectification,7 mainly at depolarized levels far from Erev.8 However, the fast kinetics of the Mg2+ block cannot account for the observed activation kinetics, because in the absence of Mg2+, inward rectifier K+ 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 K+ 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 12 13 14 15 16 17 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 K+ concentration ([K+]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 22 23 24 except for smaller outward current amplitudes than in other species.2 3 5 25 No information is currently available on the [K+]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 [K+]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) dysfunction. We report that the [K+]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 hyperpolarization-induced "inactivation" may be accounted for both by extracellular Na+ block and by external K+ depletion, possibly facilitating a polyamine-induced channel reblock; this process seems to be significantly relieved by an increase in [K+]o.
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Methods |
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Characterization of Patients
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 63.5±8.6 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 13.2±3.0 mm; normal values, <10 mm). All the patients had compensated hypertrophy (NYHA functional class II; cardiac index, 2.67±0.51 L · min-1 · m-2; ejection fraction, 0.63±0.15; LV end-diastolic pressure, 14.0±3.1 mm Hg; echocardiographic shortening fraction, 0.35±0.06) 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 Ca2+-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-µm nylon mesh to remove debris and undigested tissue. Cells were then suspended in Ca2+-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-M
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 M. 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 ±10-mV voltage
steps applied from a -70-mV holding potential and calculated as
previously described.26 Series resistance was
kept <5 M (2.26±1.04 M; n=42) and was compensated by 60% to
80%; the time constant of the capacitive current decay was 0.19±0.04
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 µs, 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 Ca2+-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
[K+]o dependence of
inward rectification kinetics, different
[K+]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 Na+ ions in the internal solution were
also expected to inhibit calcium influx through
Na+-Ca2+ exchange.
Ca2+ current was inhibited by 2 mmol/L
Co2+, Ito was
inhibited by 3 mmol/L 4-aminopyridine, and
Na+ current was minimized by the
voltage-clamp protocol.
Kinetic Analysis
The results are expressed as mean±SEM. 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
G=I/(Vm-Erev), where
Vm is membrane potential. Then, G was
normalized to its maximum value, Gmax, obtained
at Vm=-130 mV, which was 50 mV negative to
Erev:
PO=G/Gmax. Single Boltzmann
fits were used to describe the voltage dependence of
PO.
It was clear from tail currents at -130 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 -130 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[-(t-k)/
]+C, 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 -130 mV,
[C-I(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,Inst=[I(0)/C]-PO, 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 Mg2+ 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[-(t-k)/f]+As
exp[-(t-k)/s]+B, where
f and Af are the time
constant and initial amplitude of the fast component,
s 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.
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Results |
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The cell membrane was held at -50 mV (Vh),
40 mV positive to the predicted K+ equilibrium
potential (EK). Currents were elicited by 200-ms
voltage-clamp steps applied from Vh to -170 to
-20 mV at 0.16 Hz. After a 5-ms return to Vh, a
50-ms voltage-clamp pulse was delivered to -140 mV. Inward
rectification was evidenced by larger current amplitudes at voltage
steps negative to -80 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 -100 mV, and
marked declines at stronger hyperpolarizations
caused a negative slope region in the steady-state relation (Figure 1B). Erev was between -80 and -70 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 0.57±0.15 pA/pF at -50 mV.
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) and steady-state (
) current densities obtained in 16
cells bathed in 4 mmol/L [K+]o. In all
figures, SEMs are shown when they are larger than symbols.
Increasing [K+]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
[K+]o (Figure 2A). Linear fitting to mean
Erev plotted against log
[K+]o yielded a slope of
51.5±3.0 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
K+ ions. The square-root dependence of
conductance of inward rectifiers on
[K+]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
[K+]o and fitted by a
linear regression function. The mean slope of regression lines obtained
from individual cells was 0.54±0.19 (n=11).
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) mmol/L
[K+]o. A, b, Same data between -60 and -20
mV with expanded ordinates. B, Dependence of Erev on
[K+]o. Semilogarithmic plot of
Erev determined at 4, 8, and 20 mmol/L
[K+]o (n=11). Solid line indicates linear fit
to data points; dashed line, Nernst equation. C, Bilogarithmic plot of
slope conductance vs [K+]o.
"Activation" (Unblock) Kinetics
As in native and cloned channels,20 25 the
"activation" phase of current followed a
monoexponential, voltage-dependent, and
K+-sensitive time course (Figure 3). The time constant decreased as the
test potential was made more negative, whereas increasing
[K+]o caused a rightward
shift of the "activation" time constant,
act, 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
[K+]o, respectively. When
the time constants at different
[K+]o values were
replotted against Vm-EK to
account for the shift in Vrev induced by
increasing [K+]o, the
data points were superimposed along the same line (not shown).
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It is currently acknowledged that "activation" of inward currents
results from the relief of Mg2+ 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 -130 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%, 64%, and 36% of the maximum inward
current on hyperpolarization from -100, -90, and
-50 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 increased when prepulse levels
were successively depolarized from -100 to -50 mV. Conversely, the
proportion of channels blocked by the least protonated molecules,
PC,Inst, corresponds to the value
1-PC,Slow-PO, 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
[K+]o successively
applied (Figure 4B). Increasing
[K+]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
[K+]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 [K+]o,
V1/2 and EK mean values
were (in mV) -92.3, -90.4; -74.2, -72.8; and -48.5, -49.5,
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 (-90 and -73 mV at 4 and 8
mmol/L [K+]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 [K+]o but did not
cross over the PC,Slow-voltage relationship
within the voltage range studied, in contrast to previously reported
findings.6
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), -90 (
),
and -50 (
) to -130 mV, with superimposed fits (tau ranging between
1.7 and 2.0 ms for all traces). B, PO curves at 4 (
"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 -120, -130, -150, and -170
mV, the time constants of the fast and slow components,
f and s, were (in ms)
30, 243; 25, 135; 16, 53; and 11, 33, respectively.
The [K+]o dependence of
"inactivation" kinetics was investigated in cells successively
bathed in 4, 8, and 20 mmol/L
[K+]o and using voltage
steps to -170 mV (Figure 5A). As
[K+]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
[K+]o, respectively. This
behavior was observed in 5 of 5 cells studied.
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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/Imax=1/{1+exp[(V1/2-Vm)/k]}+B,
where B is the steady-state baseline level. In 4 and 8 mmol/L
[K+]o,
V1/2 (Vm for
I/Imax=0.5) and k were (in mV) -154.8±2.1,
8.1±1.1 mV and -161.9±1.3, 11.0±1.3, respectively; in 20
mmol/L [K+]o, k was
22.2±2.8 mV. For potentials more negative than -140 mV, the
availability was higher with increasing
[K+]o: the calculated
steady-state baseline level was 0.19±0.02, 0.31±0.03, and 0.60±0.05
in 4, 8, and 20 mmol/L
[K+]o, respectively.
The time-dependent decrease of IK1 during
hyperpolarizing pulses was attributed in part to extracellular
K+ 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
[K+]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 -8.2-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
K+ depletion amounted -7.0±1.3 mV (n=7).
Assuming that intracellular K+ activity does not
change, this shift in Erev corresponded to a
decrease of 1.1 mmol/L in
[K+]o (Figure 2B). From
the expected change in conductance resulting from this
[K+]o decrease (Figure 3C) and the consequent change in driving force, we may expect a current
decline of 24% at -170 mV caused by depletion of extracellular
K+ in the unstirred solution layer close to the
cellular surface. Because the difference between peak and steady state
at -170 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 -170 mV.
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Discussion |
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The major findings of the present study are threefold. (1) The nonconductive states in human ventricular IK1 channels follow a [K+]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 [K+]o may relieve the "inactivation" induced by strong hyperpolarizations.
[K+]o Dependence of
IK1 Conductance in Human Ventricular
Myocytes
The square-root dependence of conductance on
[K+]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-[K+]o
relationship may depend on several polyamine-related
factors.20 Indeed, an approximate square-root
dependence can be found in macroscopic native currents (eg, in the
present study), possibly because conductance measurements are
performed at [K+]o much
lower than the expected internal K+
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
[K+]o just reflects this
behavior.27
[K+]o Dependence of Unblocking
Kinetics
We previously showed that action potential duration (APD) is
prolonged in human LV septal cells from nonfailing hypertrophied
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 5 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 22 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
[K+]o (Figure 4). Since
the PO fitted curve reflected the voltage
dependence of inward rectification, the
[K+]o-dependent shift of
inward rectification was estimated by the
V1/2EK relationship,
which demonstrated high sensitivity on
[K+]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
[K+]o dependence of
PC,Inst and PC,Slow was
reminiscent of that established for the proportion of channels blocked
by Mg2+ (PMg) and
spermine (PSpm) in murine fibroblast
cells,6 with a positive shift of 20 mV by
increasing [K+]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 Mg2+ and putrescine
(Put2+) to increase outward currents as
follows.6 Given that Put2+
and Mg2+ blocking rate constants are larger than
that of spermine,
Put2+/Mg2+-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
Put2+/Mg2+-blocked to
spermine-blocked states, and this occurs through an intermediate
open state. As a result, the larger the number of
Mg2+/Put2+-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
[K+]o Dependence of Current
"Inactivation"
Increasing [K+]o
apparently relieves IK1 "inactivation"
during hyperpolarizing pulses (Figure 5A). The steady-state "inactivation"
shown in Figure 5B also favors the notion that high
[K+]o significantly
alleviates "inactivation," because after strong hyperpolarizing
pulses, the current availability is 3-fold greater in 20 than in
4 mmol/L [K+]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 Na+, 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 Na+ substitutes are used.25 28 29 However, this phenomenon may develop in the absence of external Na+ in guinea-pig ventricular cells31 and in Kir2.3 channels expressed in Xenopus oocytes.14 In contrast, the notion of [Na+]o block–induced "inactivation" has emerged from the use of various Na+ substitutes subsequently considered inhibitory28 or even as open-channel blockers.32 Indeed, it is unlikely that [Na+]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, Na+/K+ competition in the channel pore may be an important mechanism by which increasing [K+]o can relieve "inactivation."
Extracellular K+ 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 K+ 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 K+ conduction in IK1 channels through electrostatic interactions.20 Thus, the extracellular K+ depletion induced by hyperpolarization might reduce K+ 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 K+ in restricted spaces are expected to be less important in high than in low [K+]o, this "interactive" mechanism might also contribute to the relief of "inactivation" induced by high [K+]o together with the Na+/K+ 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 Mg2+ on
depolarization.6 Therefore, APD prolongation
usually observed in the present model30 or
slight resting potential depolarizations can reduce the proportion of
Mg2+/Put2+-blocked 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
[K+]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
K+ loss subsequent to acidification at the early
stage of acute ischemia may entail a rightward shift of
blocked-state relationships. A subsequent increased prevalence of
high-affinity polyamine-blocked states in the plateau voltage range
might be expected from the observed patterns (Figure 4), with a
resulting decrease in outward current availability and AP
prolongation.
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Acknowledgments |
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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.
Received March 16, 1998; revision received July 29, 1998; accepted August 13, 1998.
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