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(Circulation. 1995;92:3497-3504.)
© 1995 American Heart Association, Inc.

Articles

Delayed Rectifier Channels in Human Ventricular Myocytes

Marieke W. Veldkamp, PhD; Antoni C.G. van Ginneken, PhD; Tobias Opthof, PhD; Lennart N. Bouman, PhD

From the Department of Physiology (M.W.V., A.C.G.vG., L.N.B.), Academic Medical Center, and the Department of Clinical and Experimental Cardiology (T.O.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Correspondence to Marieke W. Veldkamp, Department of Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

	Abstract

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Background Previous studies have shown that in heart there are two kinetically distinct components of delayed rectifier current: a rapidly activating component (I_Kr) and a more slowly activating component (I_Ks). The presence of I_Kr and/or I_Ks appears to be species dependent. We studied the nature of the delayed rectifier current in human ventricle in whole-cell and single-channel experiments.

Methods and Results Ventricular myocytes were obtained from hearts of patients with ischemic or dilated cardiomyopathy. Single-channel currents and whole-cell tail currents were recorded at negative potentials directly after return from a depolarizing step. Single-channel currents were measured in the cell-attached patch configuration with 140 mmol/L K⁺ in the pipette. In the present study, we identified a voltage-dependent channel with a single-channel conductance of 12.9±0.8 pS (mean±SEM, n=5) and a reversal potential near to the K⁺ equilibrium potential, suggesting that the channel is selective to K⁺ ions. Channel activity was observed only after a depolarizing step and increased with the duration and amplitude of the depolarization, indicating time- and voltage-dependent activation. Activation at +30 mV was complete within 300 milliseconds, and the time constant of activation, determined in the whole-cell configuration, was 101±25 milliseconds (mean±SEM, n=4). The voltage dependence of activation could be described by a Boltzmann equation with a half-activation potential of -29.9 mV and a slope factor of 9.5 mV. The addition of the class III antiarrhythmic drug E-4031 completely blocked channel activity in one patch. No indications for the presence of I_Ks were found in these experiments.

Conclusions The conformity between the properties of I_Kr and those of the K⁺ channel in the present study strongly suggests that I_Kr is present in human ventricle.

Key Words: ventricles • potassium • delayed rectifyer current

	Introduction

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A delicate balance between small inward and outward currents exists during the plateau phase of the ventricular action potential. Activation of the delayed rectifier current (I_K) initiates the onset of final repolarization. Consequently, the kinetics of this current are an important parameter for action potential duration and refractoriness.^{1 2 3 4} The delayed rectifier current has gained a lot of interest because blockade of this current by specific class III antiarrhythmic agents may provide a tool for action potential prolongation with concomitant increase in refractory period,^{5 6 7} which is considered to have an antiarrhythmic effect.⁸ Voltage-clamp experiments with the class III antiarrhythmic drug E-4031 have revealed the existence of two kinetically and pharmacologically distinct types of delayed rectifier current: a rapidly activating component (I_Kr), which is blocked by E-4031, and a more slowly activating component (I_Ks), which is insensitive to E-4031.^{9 10} Furthermore, I_Kr activates at relatively negative potentials and shows inward rectification at positive potentials, whereas I_Ks activates at more positive potentials and has a linear current-voltage relation. The presence of I_Kr and I_Ks appears to be species dependent. Although in some species these two types coexist,^{1 9 10 11 12} other species display either I_Ks^{13 14} or I_Kr.^{15 16 17 18 19 20 21} The aim of the present study was to investigate at the single-channel level the type of I_K that is present in isolated human ventricular myocytes. Data on I_K in human heart are scarce. Recently, it was shown that I_K is present in both human atrial²² and ventricular²³ myocytes, although in ventricle, I_K was very small and could be identified in only a minority of the cells. The observation that I_K in atrial myocytes is partially blocked by E-4031 demonstrates that both I_Kr and I_Ks are present in human atrium.²⁴ Whether I_K in human ventricle consists of one or two components has not been elucidated. Results of the present study show for the first time measurements of the delayed rectifier current at the single-channel level in human ventricle. It appears that this current is comparable to I_Kr in other mammalian species.

	Methods

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Preparation of Single Ventricular Myocytes
Ventricular myocytes were isolated from explanted human hearts by an enzymatic dissociation procedure. The hearts were obtained from nine patients with end-stage heart failure due to ischemic cardiomyopathy (n=3) or dilated cardiomyopathy (n=6) who were undergoing heart transplantation. Informed consent was obtained before heart transplantation. Directly after explantation, the heart was transported to the laboratory in oxygenated Tyrode's solution (4°C). A portion of the left ventricular free wall was dissected and mounted on a Langendorff perfusion apparatus, where it was perfused through a branch of the left anterior descending coronary artery with solutions in the following sequence: (1) normal Tyrode's solution (10 minutes), (2) Ca²⁺-free Tyrode's solution (10 minutes), and (3) Ca²⁺-free Tyrode's solution to which collagenase type B (0.1 to 0.25 mg/mL, Boehringer Mannheim), collagenase type P (0.1 mg/mL, Boehringer Mannheim), and trypsin inhibitor (0.15 mg/mL, Boehringer Mannheim) had been added (20 to 30 minutes). Portions of the left ventricular wall that were clearly digested by the enzymes were cut into small pieces that were gently agitated in a small beaker with Kraft-Brühe (KB)²⁵ solution to obtain single cells. Tissue pieces were taken throughout the ventricular wall, so cell suspension contained cells from epicardial, midcardial, and endocardial origins. During the entire isolation procedure, the solutions were oxygenated and temperature was maintained at 37°C. Cells were allowed to sediment for 15 minutes, after which half of the supernatant was replaced by normal Tyrode's solution to increase the extracellular Ca²⁺ concentration. This procedure was repeated three times before the experiments.

Solutions and Reagents
The composition of the solutions was as follows (in mmol/L): normal Tyrode's solution—NaCl 140, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, glucose 5.5, and HEPES 5.0, pH adjusted to 7.4 with NaOH; Ca²⁺-free Tyrode's solution—NaCl 140, KCl 5.4, MgCl₂ 0.5, KH₂PO₄ 1.2, glucose 5.5, and HEPES 5.0, pH adjusted to 6.9 with NaOH; and KB solution—KCl 85, K₂HPO₄ 30, MgSO₄ 5.0, glucose 20, pyruvic acid 5.0, creatine 5.0, taurine 5.0, EGTA 0.5, ß-hydroxybutyric acid 5.0, succinic acid 5.0, Na₂ · ATP 2.0 and 50 g/L polyvinyl pyrrolidone · 40, pH adjusted to 6.9 with KOH. Pipette solution for whole-cell recording of I_K tail currents contained (in mmol/L) KCl 140, K₂ATP 5.0, and HEPES 10, pH adjusted to 7.2 with KOH. Pipette solution for whole-cell recording of action potentials contained (in mmol/L) KCl 140, and HEPES 10, pH adjusted to 7.2 with KOH. Pipette solution for cell-attached patch recording of single-channel currents contained (in mmol/L) KCl 140, CaCl₂ 2.0, MgCl₂ 1.0, and HEPES 10, pH adjusted to 7.4 with KOH. E-4031 (Eisai) was prepared as a 1-mmol/L stock solution.

Recording Procedures
Small aliquots of cell suspension were introduced to a cell chamber placed on the temperature-controlled stage of an inverted microscope (Nikon Diaphot). The isolated myocytes were allowed to adhere to the bottom of the cell chamber for 3 minutes, after which perfusion with normal Tyrode's solution was started. The cells were continuously superfused with normal Tyrode's solution at a rate of 1 mL/min. The temperature of the bath was maintained at 34±2°C. Currents were recorded in the whole-cell or cell-attached patch configuration of the patch-clamp technique²⁶ with a laboratory-made patch-clamp amplifier. The pipettes were pulled from borosilicate glass by a laboratory-made one-stage puller. The tips of the pipettes were heat polished. After filling with the appropiate pipette solution, the pipettes had a resistance of 3 to 5 M.

Action potentials were elicited at 0.5 Hz (0.44 to 0.65 Hz) at a stimulus strength of 1.5 times diastolic threshold. Action potential duration was determined with a device that measured the time when the membrane potential was above an adjustable trigger level. The level was set to 95% of the amplitude of the action potential. The voltage-clamp protocol for channel activation consisted of depolarizing steps of variable amplitude and duration at 1.5- to 3.5-second intervals. Single-channel and whole-cell tail currents were measured at negative membrane potentials directly after return from the depolarizing step. With 140 mM K⁺ in the pipette, single-channel currents were recorded as inward currents, and channel openings are plotted as downward deflections in the recordings. The patch membrane potential was obtained as the difference between an average resting membrane potential of -70 mV (-71.2±1.14 [mean±SEM], n=15) and the patch pipette potential. Whole-cell and single-channel currents were stored on digital audio tape with the use of a digital tape recorder (DTR 1200, Biologic). Membrane currents were off-line filtered (low-pass) at 0.5 kHz (single-channel currents) or 2 kHz (whole-cell currents) with a two-pole Butterworth filter, digitized at a sampling interval of 667 µsec with an AD converter board (National Instruments), and stored on computer hard disk (Apple Macintosh) for subsequent data analysis.

Data Analysis
Single-channel recordings were corrected for leakage current and capacitive transients and evaluated with the use of a laboratory-made analysis program. Correction for capacitive transients was accomplished by subtraction of an exponential function. The time constant and amplitude of the exponential function were adjusted until a flat baseline was obtained. Whole-cell currents were not compensated for capacitance and series resistance. The series resistance varied between 6.2 and 13 M.

In whole-cell experiments, the degree of activation was determined by measuring the amplitude of the tail current at a fixed potential (-50 mV), directly after a depolarizing step. Tail current amplitude was defined as the difference between the peak current and the steady state current at -50 mV. The amplitude of the tail current is considered to be proportional to the degree of activation during the preceding depolarization. Analogous to this, to estimate the degree of activation in single-channel experiments, one may construct ensemble average currents and measure peak amplitudes. However, the construction of ensemble averaged currents was hampered by the presence of inward rectifier channel activity. Therefore, we estimated the degree of activation in the following, alternative way:

When the voltage, at which current tails are measured, is constant, one may also expect that the time constant of current decay is constant. Therefore, the area-under-the-current tails is also proportional to the degree of activation. The area-under-the-tail current is given by:

where A is the area-under-the-tail current, ñ_open is the mean number of channels open during time T, and i is the single-channel current amplitude. In the experiments presented here, T was the first 800 ms after return to -70 mV. Because T is constant and i is constant (at one particular voltage), ñ_open is directly proportional to A and thus to the degree of activation. Consequently, we used ñ_open as a measure for the degree of activation in the single-channel experiments.

The mean number of open channels in the patch membrane was derived from amplitude histograms constructed from several traces. The area under each peak in the amplitude histogram, representing the fraction of time that 0, 1, 2, ..., n channels are simultaneously in the open state, was calculated from gaussian functions fitted through these peaks. The height, width, and mean of the gaussian functions were adjusted to the data by the eye. The mean number of open channels was then calculated in the following way:

where n is the number of channels simultaneously in the open state, n_max is the total number of channels in the patch membrane, and P_n is the fraction of time that n channels are simultaneously in the open state.

	Results

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Fig 1 shows an action potential recorded in a cell from a patient with dilated cardiomyopathy at a 1870-ms pacing cycle length. The action potential configuration is characterized by a prominent notch during phase 1 and a long-lasting plateau phase. The action potential duration was 1120 milliseconds (ms). In a total of five cells driven at 0.5 Hz (0.44 to 0.65 Hz), action potential duration was 1247±32 ms (mean±SEM), substantially longer than reported in normal human single ventricular cells.²³

View larger version (6K): [in this window] [in a new window]   Figure 1. Action potential recorded in a ventricular myocyte isolated from the heart of a patient with dilated cardiomyopathy. Stimulus frequency was 0.54 Hz, and action potential duration was 1120 milliseconds (ms).

Depolarization Activates a 12.4-pS K⁺ Channel
Fig 2 shows a typical example of membrane currents recorded at -70 mV from a cell-attached patch of a human ventricular myocyte. Most patches contained inward rectifier (I_Kl) channels, identified by openings of long duration and a single-channel conductance of 41.5 pS (see Fig 4). This value is close to the values reported for I_Kl channels in rabbit ventricle^{27 28 29} but somewhat larger than values reported for I_Kl channels in guinea pig ventricle.^{30 31 32}

View larger version (15K): [in this window] [in a new window]   Figure 2. A small conductance channel is activated by depolarization in human ventricular cell membrane. Top trace, Voltage protocol. Bottom traces, Single-channel currents recorded at -70 mV from a cell-attached patch of a human ventricular myocyte before and directly after a depolarizing step (500 ms) to +30 mV. Before depolarization, only inward rectifier channel activity is observed. Directly after a depolarizing step, channel openings of smaller amplitude are also seen (arrows). Channel openings are seen as downward deflections. (Origin myocyte, ischemic cardiomyopathy.)

View larger version (25K): [in this window] [in a new window]   Figure 4. Current-voltage relation of the human delayed rectifier channel and the inward rectifier channel. A, Single-channel activity recorded from a cell-attached patch at various negative membrane potentials after return from a depolarizing step (500 ms) to +30 mV. Channel openings are seen as downward deflections. Solid lines indicate the zero current level; dashed lines, unitary current level of the open inward rectifier channel; and dotted lines, unitary current level of the open delayed rectifier channel. (Origin myocyte, dilated cardiomyopathy.) B, Voltage protocol. C, Graph showing the unitary current amplitudes of the delayed rectifier () and inward rectifier () channel plotted against the applied membrane potential for data from five patches. Data from each experiment were fitted separately by linear regression. Straight line through the pooled data represents a linear function with values for slope conductance and extrapolated reversal potential that are the mean of five experiments. The slope conductance was 12.9±0.8 and 41.5±3.8 pS for the delayed rectifier and inward rectifier channel, respectively. The extrapolated reversal potential was -15±7 and -16±5 mV for the delayed rectifier and inward rectifier channel, respectively.

To activate delayed rectifier (I_K) channels, the patch membrane was depolarized from -70 mV to +30 mV for 500 ms (Fig 2). During the depolarization to +30 mV, no channel activity could be observed. However, after repolarization to -70 mV, a series of short channel openings was seen, superimposed on either the open or closed state of the I_Kl channel. The unitary current amplitude was approximately one third of that of the I_Kl channel. Unlike I_Kl channel activity, this type of channel activity disappeared shortly after repolarization and could be evoked again only by another depolarizing step. In 6 of 26 ptaches (23%), we observed this type of channel activity. Although channel openings could not be observed in any of these patches during depolarization, these data suggest that this channel is activated during depolarization and that the decay of channel activity seen after return to -70 mV is due to deactivation.

Fig 3A shows another example of this type of channel activity recorded at -70 mV after a depolarizing step to +30 mV. In this experiment, the patch was exposed to 10^-7 mol/L E-4031, a class III antiarrhythmic drug that has been shown to specifically block a component of the delayed rectifier current. Approximately 1 minute after the superfusion with E-4031, channel openings occurred less frequently (Fig 3B) and disappeared completely after another minute (Fig 3C). Washout did not restore channel activity within the duration of the experiment. Because channel activity was not restored after washout of E-4031, one might argue that rundown is involved. However, in none of our single-channel experiments was a significant decrease in channel activity observed during the experiment. In this particular experiment, channel activity before the addition of E-4031 had been stable for almost 1 hour, and it is unlikely that channel activity started to run down exactly at the moment of addition of the drug. Finally, the time needed for the drug to block the channels after switching solutions was comparable to the time needed to block I_K channels in rabbit ventricular myocytes at the same perfusion rate.²⁹ We refer to this channel as an I_K channel.

View larger version (18K): [in this window] [in a new window]   Figure 3. The class III antiarrhythmic drug E-4031 blocks the small conductance channel. Single-channel currents recorded from a cell-attached patch at -70 mV directly after return from a depolarizing step (duration, 500 ms) to +30 mV. In this patch, no inward rectifier channel activity was observed. Channel openings are seen as downward deflections. Top trace, Voltage protocol. A, Consecutive tracings recorded during control conditions. B, Consecutive tracings recorded after 1-minute perfusion with 10-7 mol/L E-4031. C, Consecutive tracings recorded after 2-minute perfusion with 10-7 mol/L E-4031. (Origin myocyte, dilated cardiomyopathy.)

To determine the single-channel conductance of this channel, we depolarized the patch membrane to +30 mV and measured the unitary current amplitude after repolarization to various test potentials (Fig 4). The unitary current amplitude of both the I_Kl channel and the I_K channel increased with hyperpolarization (Fig 4A). The decay of I_K channel activity was much faster at -110 mV than at more depolarized potentials (eg, -70 mV), suggesting a voltage dependence of deactivation. Fig 4C shows the current-voltage relation of both channels. When the data from five experiments were fit by linear regression, a slope conductance of 12.9±0.8 pS for the I_K channel and 41.5±3.8 pS for the I_Kl channel was determined. Extrapolation of the current-voltage curve yielded an estimated reversal potential of -15±7 mV for the I_K channel, which is near the K⁺ equilibrium potential (0 mV with 140 mmol/L K⁺ in the patch pipette). This result suggests that the investigated channel is selective to K⁺ ions.

Time Course of Activation: Single-Channel Analysis
The time course of activation of the human I_K channel was studied by depolarizing cell-attached patches to +30 mV for various durations and by measuring single-channel activity after return to -70 mV (as a measure for the degree of activation). The single-channel currents (Fig 5, left) were recorded from a cell-attached patch containing one I_Kl and one I_K channel. Fig 5 (right) shows the accompanying amplitude histograms. In this experiment, a 50-ms lasting depolarization was too short to elicit I_K channel activity (Fig 5, top left). After repolarization to -70 mV, only I_Kl channel activity was observed, which resulted in two peaks in the amplitude histogram. One peak corresponds to the closed I_Kl channel (0 pA), and one peak corresponds to the open I_Kl channel (-2.1 pA). After return to -70 mV from a 100-ms lasting depolarization, some traces contained I_K channel activity, indicated by two extra peaks in the amplitude histogram (Fig 5, right, arrows). The peak at -0.7 pA corresponds to the open I_K channel when the I_Kl channel is closed, and the peak at -2.7 pA corresponds to the open I_K channel when the I_Kl channel is also open. Lengthening of the depolarizing pulse to 200 ms resulted in a further increase in the number of traces containing I_K channel activity. This increase in I_K channel activity can be appreciated by the increase in the area under the peaks corresponding to the open I_K channel in the amplitude histogram (Fig 5, right). Depolarizations of 300 ms in duration did not result in a further increase in I_K channel activity. To determine the degree of activation during depolarization, the mean number of open channels (ñ_open) during the first 800 ms after return to -70 mV was calculated from the amplitude histograms (see Methods). Values for ñ_open were normalized to the maximum value (ñ_open,max) and were plotted versus the duration of the depolarization (Fig 6). The time dependence of activation shows that the increase in ñ_open saturates for depolarizations of 250 ms and longer, indicating that activation of the I_K channel is complete within this length of time. Similar results were obtained in an additional two patches. It was not possible to determine the time constant of activation for these single-channel data. Due to the limited number of events, there is considerable scatter in the ñ_open values, which makes it difficult to reliably fit the data points with an exponential function.

View larger version (26K): [in this window] [in a new window]   Figure 5. Delayed rectifier channel activity is increased after depolarizations of increasing duration. Top, Voltage protocol. Left, Typical examples of single-channel currents recorded from a cell-attached patch at -70 mV after depolarizing steps to +30 mV of various duration. Stimulation interval was 2.5 seconds. Channel openings are seen as downward deflections. Solid lines indicate the zero current level; dashed lines, unitary current level of the open inward rectifier channel; and dotted lines, unitary current level of the open delayed rectifier channel. Right, Amplitude histograms constructed from 12 to 14 traces of 800 ms in duration. The peaks in the amplitude histograms corresponding to the open IK channel are indicated with arrows. In this particular experiment, there also was an increase in IKl channel activity on longer depolarizations. This was, however, a solitary observation. (Origin myocyte, ischemic cardiomyopathy.)

View larger version (11K): [in this window] [in a new window]   Figure 6. Plot of time dependence of activation at +30 mV. ñopen indicates mean number of open channels; ñopen,max, maximum value of ñopen. The normalized ñopen values during the first 800 ms after return to -70 mV (from experiment shown in Fig 5) are plotted against the duration of the preceding depolarization to +30 mV.

Time Course of Activation: Whole-Cell Tail Current Analysis
To determine the time course of activation of the macroscopic I_K, whole-cell tail currents were studied. In 9 of 12 cells (75%), small, slowly decaying tail currents were observed after return from a depolarizing step. In 4 of these cells, tail current analysis was performed. Fig 7A shows superimposed whole-cell current traces obtained during depolarizations from -50 mV to +30 mV of various durations and subsequent tail currents (I_K,tail) after return to -50 mV. No measures were taken to suppress other voltage-dependent currents active at depolarized potentials. Increasing the duration of the depolarizing step clearly increased I_K,tail amplitude up to depolarizations of 300 ms in duration (Fig 7B). When the normalized tail current amplitude was plotted versus the duration of the depolarization (Fig 7C), data points could be fitted with a monoexponential function of the form:

View larger version (12K): [in this window] [in a new window]   Figure 7. Time course of activation of the whole-cell delayed rectifier current. A, Voltage protocol and superimposed current traces when the membrane was depolarized from -50 to +30 mV for various duration and subsequent tail currents after return to -50 mV. The stimulation interval was 2 seconds. Dashed line indicates zero current level. B, Enlarged tail currents of A. C, Plotted are the normalized tail current amplitudes against the duration of the depolarization. Solid line represents the best fit of a single exponential function. The fit was allowed to approach the asymptote with a self-chosen maximum A (0.95). The time constant of activation was 140 ms (R=.98). (Origin myocyte, dilated cardiomyopathy.)

where A is a free parameter and represents the maximum value of the exponential function, t is the duration of the depolarizing step, and is the time constant of activation at this potential (+30 mV). For this particular experiment, was 140 ms. This type of experiment was performed in a total of four cells, and the average time constant of activation was 101±25 ms (mean±SEM).

Voltage Dependence of Activation
Cell-attached patches were depolarized for 500 ms to various potentials and channel activity after return to -70 mV was measured to determine the voltage dependence of activation (Fig 8). The single-channel currents shown in Fig 8 (left) were recorded from a cell-attached patch containing one I_Kl channel and one I_K channel. Fig 8 (right) shows the accompanying amplitude histograms. After return to -70 mV from a depolarization to -50 mV, only a few traces displayed I_K channel activity (Fig 8, top left). The very small peak indicated by an arrow in the amplitude histogram corresponds to one open I_K channel when the I_Kl channel is also open (Fig 8, top right). Depolarization to -20 mV clearly increased I_K channel activity, as expressed by the two peaks in the amplitude histogram. One peak corresponds to the open I_K channel when the I_Kl channel is closed, and one peak corresponds to the open I_K channel when the I_Kl channel is also open. Depolarization to +10 mV resulted in a further increase in I_K channel activity, as may be appreciated from the increase in the area under the peaks corresponding to the open I_K channel. Thus, I_K channel activity increases when the preceding depolarizing step is more positive.

View larger version (29K): [in this window] [in a new window]   Figure 8. Delayed rectifier channel activity is increased after depolarizations of increasing amplitude. Top, Voltage protocol. Left, Typical examples of single-channel currents recorded from a cell-attached patch at -70 mV after depolarizing steps to various potentials (duration, 500 ms). Stimulation interval was 3 seconds. Channel openings are seen as downward deflections. Solid lines indicate the zero current level; dashed lines, unitary current level of the open inward rectifier channel; and dotted lines, unitary current level of the open delayed rectifier channel. Right, Amplitude histograms constructed from 10 or 11 traces of 800 ms in duration. The peaks in the amplitude histogram corresponding to the open IK channel are indicated with arrows. (Origin myocyte, dilated cardiomyopathy.)

The ñ_open during the first 800 ms after return to the resting membrane potential was calculated from the amplitude histograms and was used as a measure for the degree of activation during the preceding depolarization. Normalized ñ_open values were plotted versus the potential during depolarization (Fig 9). The data points were fitted by a Boltzmann equation:

View larger version (11K): [in this window] [in a new window]   Figure 9. Plot of voltage dependence of activation. ñopen indicates mean number of open channels; ñopen,max, maximum value of ñopen. The normalized ñopen values during the first 800 ms after return to -70 mV (from experiment in Fig 7) are plotted against the patch potential during the preceding depolarization (500 ms). Solid line represents the least-squares fit of a Boltzmann relation with a half-activation potential of -29.9 mV, a slope factor of 9.5 mV, and a self-chosen maximum C of 0.93 (R=.98).

where C is a free parameter and represents the maximum value of the Boltzmann function, V_1/2 is the half-activation potential, V_m is the membrane potential, and K is the slope factor. We found a V_1/2 of -29.9 mV and a K of 9.5 mV.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we describe for the first time measurements of single-channel currents through native delayed rectifier channels in the membrane of human ventricular myocytes. The investigated channel was identified as a delayed rectifier channel on the basis of the following observations: (1) channel activity was observed only after return from a depolarizing step and increased with the duration of the depolarizing step, indicating time-dependent activation on depolarization (Figs 2 and 5); (2) the channel had a slope conductance of 12.9±0.8 pS (mean±SEM) and an (extrapolated) reversal potential of -15±7 mV, which is close to the expected K⁺ equilibrium potential and suggests that the channel is highly permeable for K⁺ ions (Fig 4); (3) increasing the amplitude of the depolarizing step increased channel activity, indicating voltage dependence of activation (Figs 8 and 9); and (4) after return from a depolarizing step, channel activity recorded at negative membrane potentials decayed with time, indicating deactivation upon repolarization (Figs 2 and 4).

Comparison to Rapidly Activating Delayed Rectifier Currents in Other Species
In several species, the cardiac delayed rectifier current is composed of a slowly activating component (I_Ks) and a more rapidly activating component (I_Kr), with different kinetic, conductive, and pharmacological properties. I_Kr activates at more negative potentials than I_Ks and displays marked inward-going rectification, whereas I_Ks has a linear current-voltage relation. I_Kr is specifically blocked by the class III antiarrhythmic agent E-4031, and I_Ks is not. The presence of I_Kr and/or I_Ks appears to depend on species. I_Kr is the only component in cat ventricular^{19 20 21} and rabbit sinoatrial, atrioventricular, and Purkinje fiber myocytes,^{15 16 17 18} whereas I_Ks is the only component in frog atrial myocytes.^{13 14} Both components have been identified in myocytes from guinea pig atrium and ventricle,^{9 10 12} sheep Purkinje fiber,¹ and chick atrium.¹¹ Recently, the presence of both components has been demonstrated in human atrium.²⁴

The rapid time course of activation found in the present study strongly suggests that the I_K channel in human ventricle resembles I_Kr. Activation at +30 mV was complete within 300 ms, and the time constant of activation, obtained by whole-cell tail current analysis, was 101±25 ms (mean±SEM). This value compares well with the activation time constants at comparable voltages reported for I_Kr in human atrium (100 ms),²⁴ mouse atrial tumor myocytes (120 ms),³³ and a rapidly activating I_K channel we previously identified in rabbit ventricle (187 ms)²⁹ but are slower than the activation time constants for I_Kr in guinea pig (20 ms)^{9 10} and rabbit sinoatrial node (40 ms).¹⁶

Other characteristics of this human I_K channel are also consistent with those of I_Kr. The single-channel conductance of 12.9 pS is close to the conductances found for I_K channels with relatively rapid kinetics in rabbit sinoatrial node (11.1 pS)¹⁶ and ventricle (13.7 pS)²⁹ and guinea pig atrium (10 pS)³⁴ assessed under similar conditions.

I_K channel activity could be evoked at voltages positive to -60 mV and saturated for depolarizations to 0 mV (Fig 8). Therefore, the I_K channel in the present study shows a similar voltage dependence as I_Kr. Although slightly more negative, the half-maximal activation potential in human ventricle (-29.9 mV in the present study) compares with that for I_Kr in rabbit sinus node (-25.1 mV)¹⁶ and guinea pig atrium and ventricle (-19.3 and -21.5 mV).^{9 10} Also, the slope factor (9.5 mV in the present study) was close to that found in the other studies (ranging from 5.2 to 7.5 mV).^{9 10 16} The fact that we failed to observe channel openings during depolarizations at positive potentials may point to inward rectifying properties of this channel, which is also characteristic of I_Kr.^{9 10 16 24 35} Finally, the observation that channel activity was completely blocked by E-4031 also favors a resemblance to I_Kr.

In the past few years, a number of voltage-gated K⁺ channels have been cloned from the cardiovascular systems of rat and humans.^{36 37 38} These cloned channels can approximately be divided into two groups: (1) channels with fast activation and fast inactivation kinetics and (2) channels with fast activation kinetics and no or little, slow inactivation. The latter group consists of delayed rectifier-type channels, but none of these channels resembles the channel presented in the present study because the activation time course of these cloned channels is at least one order of magnitude faster than the human I_K channel in this study.

In our experiments, we found no indication of the presence of I_Ks at the single-channel level. It has been reported, however, that the single-channel conductance of I_Ks is very small (<1 pS).³⁹ Therefore, it is conceivable that I_Ks is present in human ventricle but remains unnoticed in single-channel recordings. Also, no observations were made in the whole-cell recordings that pointed to the presence of I_Ks. When I_Ks contributes significantly to membrane current, this current should have been recognized in whole-cell currents. However, the outward current during depolarization was very small (Fig 6A), even at longer depolarizations (900 ms). Moreover, the increase in whole-cell tail current amplitude saturated for depolarizations of 300 ms in duration. These observations suggest that I_Ks in human ventricle is small or absent. Similar findings were obtained in the whole-cell voltage clamp study of Beuckelmann et al.²³ They showed that in human ventricle, I_K is small (or absent) and is completely activated within 250 ms, suggesting the presence of an I_K current different from I_Ks.

It must be noted, however, that the ventricular myocytes in this study had extremely long action potentials (Fig 1), even for cardiomyopathy. The low stimulus frequency is certainly one of the causes of prolonged action potential duration. The experimental temperature was approximately 3°C below the physiological temperature and is another factor for action potential prolongation. In addition, repolarization in these cells may have been altered by the isolation procedure. Therefore, it cannot be excluded that the absence of the slow component of I_K may be due to the diseased state of the cells or to altered membrane properties caused by the isolation procedure rather than true absence of this component. Also, the possibility of rundown of I_Ks channel activity in the whole-cell configuration cannot be ignored. Therefore, we cannot exclude a role for I_Ks in the nondiseased human ventricle.

	Acknowledgments

 
The authors thank Jacques de Bakker, Jessica Vermeulen, Ton Baartscheer, and Cees Schumacher for their help in the preparation of the cells. The hearts were made available by the Department of Cardiology, A2U, Utrecht.

Received February 27, 1995; revision received July 20, 1995; accepted July 23, 1995.

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