Characterization of a Slowly Inactivating Outward Current in Adult Mouse Ventricular Myocytes

From the Cardiovascular Research Division (J.Z., A.J., X.H., G.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, and the Division of Cardiology (B.L.), University of Pittsburgh Medical Center, Pa.

Correspondence to Gideon Koren, MD, Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115. E-mail koren{at}calvin.bwh.harvard.edu

	Abstract

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Abstract—We recently have reported that suppression of the slowly inactivating component of the outward current, I_slow, in ventricular myocytes of transgenic mice (long QT mice) overexpressing the N-terminal fragment and S1 segment of Kv1.1 resulted in a significant prolongation of action potential duration and the QT interval. Here we describe the detailed biophysical properties and physiological role of I_slow by applying the whole-cell patch-clamp technique at both room temperature and 37°C. This current activates rapidly with time constants ranging from 3.8±0.8 ms at –20 mV to 2.1±0.5 ms at 50 mV at room temperature. The half-activation voltage and slope factor are –12.5±2.6 mV and 7.7±1.0 mV, respectively. The inactivation of this current is slow compared with the fast inactivating component I_to, with time constants of 100 ms at 37°C. The steady-state inactivation of I_slow is not temperature-dependent, with half-inactivation voltages and slope factors of –35.1±1.3 and –5.4±0.4 mV at 37°C, and –37.6±1.8 and –5.8±0.6 mV at room temperature. Double exponentials were required to describe the time-dependent recovery of I_slow from steady-state inactivation, with time constants of 233±34 and 3730±702 ms at 37°C, and 830±240 and 8680±2410 ms at room temperature. I_slow is highly sensitive to 4-aminopyridine but is insensitive to tetraethylammonium, -dendrotoxin, and E-4031. Stimulation with action-potential waveforms under voltage-clamp mode revealed that this current plays an important role in the early and middle phases of repolarization of the cardiac action potential. We conclude that the biophysical properties and pharmacological profiles of I_slow are similar to those of Kv1.5-encoded currents.

Key Words: K⁺ channel • electrophysiology • mouse • heart • cardiac arrhythmia

	Introduction

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Outward currents in the heart are responsible for repolarization of membrane potential and therefore influence action potential duration (APD) and electrical activity. Expression of different outward potassium currents in the heart varies among different species. The transient outward current (I_to) has been described in cat, dog, ferret, rabbit, rat, human ventricular cells, and rabbit nodal and crista terminalis cells, whereas guinea pig and frog ventricular myocytes have virtually no I_to (for review, see Giles et al¹ ). In some preparations,^{2 3 4 5} 2 components of I_to have been described: I_to1, a Ca²⁺-dependent and 4-aminopyridine (4-AP)–insensitive outward current, and I_to2, a Ca²⁺-independent and 4-AP–sensitive outward current. Several recent studies^{6 7} indicate that the charge carrier of I_to1 is Cl^-, rather than K⁺. In all the preparations, I_to2 shares similar biophysical and pharmacological properties, including rapid activation and inactivation, K⁺ selectivity, and 4-AP-sensitivity.

With increasing genetic manipulations applied to the mice, it is of crucial importance to understand the basic physiology of this animal. The electrophysiology and biophysical properties of the ionic currents expressed in the mouse heart, however, are not fully characterized. The outward current of adult mouse ventricular myocytes has been noticed to have an incomplete inactivation phase characterized by a sum of a pedestal and 2 exponentials with a fast time constant similar to that of classic I_to and a slow time constant.^{8 9 10 11 12} Very recently, Fiset et al¹³ found that the blockade of this slow inactivation current by a low concentration (50 µmol/L) of 4-AP markedly prolonged APD and exerted an inotropic effect. However, no comprehensive study of the electrophysiological features of this slowly inactivating current has been reported.

We recently have established a transgenic mouse model with a long-QT phenotype (LQT mouse)¹¹ by overexpressing a truncated Kv1.1 channel gene, Kv1.1N206, that contains the N-terminus and first transmembrane segment of Kv1.1. Electrophysiological study of the myocytes isolated from LQT mice, compared with controls, revealed prolonged APD because of a significant reduction in the density of a rapidly activating, slowly inactivating, and highly 4-AP–sensitive outward K⁺ current (I_slow). The purpose of this investigation is to describe the detailed biophysical and pharmacological properties of this current and provide further evidence that the slowly inactivating component of the outward current of mouse myocytes is likely encoded by Kv1.5.

	Materials and Methods

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Preparation of Isolated Myocytes
Control and age-matched LQT adult FVB mouse (20 to 30 g bw) hearts were removed rapidly and perfused retrogradely at 37°C (1.5 to 2 mL/min) with a oxygenated, nominally Ca²⁺-free Tyrode's solution [137 mmol/L NaCl, 5.4 mmol/L KCl, 1.0 mmol/L MgCl₂, 0.33 mmol/L NaH₂PO₄, 10 mmol/L glucose, 10 mmol/L HEPES, pH 7.35] for 3 to 4 minutes, followed by a perfusion of 4 to 6 minutes with enzyme solution [0.5 mg/mL collagenase (type I), 0.05 mg/mL protease (type XXIV, Sigma Chemical Co) and 1 mg/mL fatty acid–free BSA in the Ca²⁺-free Tyrode's solution]. The ventricles were then chopped into small pieces, incubated at 37°C in the same enzyme solution for 5 to 10 minutes, and mechanically dispersed. After repeatedly washing by a series of centrifugations at 500 rpm for 2 to 3 minutes and resuspensions in 250 µmol/L Ca²⁺-containing Tyrode's solution supplemented with 2% FCS, cells were plated on glass cover slips coated with laminin (Collaborative Research Inc) and maintained at room temperature in normal Tyrode's solution (1.0 mmol/L Ca²⁺) supplemented with 2% FCS. Calcium-tolerant, rod-shaped ventricular myocytes with clear striations were selected randomly for the electrophysiological studies.

Electrophysiological Study
All current recordings were obtained in the whole-cell, voltage-clamp configuration of the patch-clamp technique¹⁴ by using 1.2-mm OD borosilicate glass electrodes (World Precision Instruments). Most of the data presented in this study were obtained with electrodes having a resistance of 0.5 to 2 M when filled with a standard pipette solution (130 mmol/L KCl, 1 mmol/L MgCl₂, 0.5 mmol/L CaCl₂, 10 mmol/L HEPES, 5 mmol/L EGTA, 5 mmol/L Mg₂ATP, 5 mmol/L Na-creatine phosphate, and 0.5 mmol/L GTP-tris; pH 7.2 with KOH). In some parts of the study, work was performed with the internal solution, which contained 110 mmol/L K-Aspartate and 20 mmol/L KCl (all other chemicals remained the same). In this case, a junction potential of about 10 mV (10 to 13 mV, pipette negative) was corrected off-line. The electrodes were connected to an Axopatch 200A amplifier (Axon Instruments), and a DigiData 1200 (Axon Instruments) interface controlled by pClamp 6.0.4 software (Axon Instruments) was used to generate command pulses and acquire data. After formation of a high-resistance seal (5 to 40 G) between the recording electrode and the myocyte membrane, electrode capacitance was fully compensated electronically before breaking the membrane patch.

In the whole-cell configuration, the series resistance (R_s) was estimated from the decay of uncompensated capacitative transients by dividing the time constants by the calculated membrane capacitance, which was measured as the time-integral of the capacitative surge in response to 10 mV hyperpolarizing steps from a holding potential (HP) of –40 mV. In this study, cell capacitances in control and LQT myocytes studied were 166.1±8.9 (n=26) and 174.8±13.2 pF (n=20), respectively (P>0.05). The calculated R_s were within the range of 2 to 10 M, 2 to 6 times those of the pipette resistance. In most of the experiments, the cell capacitances could not be compensated fully, because the maximal capacity of Axopatch 200A is 100 pF. By adjusting R_s compensation, the capacitative transient could be limited within 1.5 ms in most of the experiments. For the recordings on large cells (eg, 150 pF), which often expressed large currents, only the electrodes that had low resistances (0.5 to 1.0 M) were used. These efforts enabled us to minimize the voltage errors (typically not >8 mV) across the electrodes after R_s compensation, and these were not corrected. In all voltage-clamp experiments, R_s compensation was checked regularly to ensure that there were no variations with time. Data were discarded if an increase in R_s was evident during the course of an experiment. All current recordings used for this study (including those presented here) are from raw records. Linear "leakage" currents were not corrected, because they were negligible in cells with input resistances 1 G.

Cells were superfused at 1 to 2 mL/min with Tyrode's solution (137 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L MgCl₂, 0.33 mmol/L NaH₂PO₄, 1 mmol/L CaCl₂, 10 mmol/L glucose, and 10 mmol/L HEPES, pH 7.4 with NaOH). Action potentials were recorded in current-clamp mode in normal Tyrode's solution by injecting suprathreshold current pulses through the patch-clamp electrode. To record the depolarization-activated K currents, 2 mmol/L CoCl₂ was used to inhibit I_Ca. Mg₂ATP in the pipettes suppressed the ATP-sensitive potassium current. Na currents were suppressed by holding the cells at –50 mV and, in some experiments, by applying a brief (15 to 20 ms) prepulse (from HP to –20 mV) before the testing steps. All recordings were started after 5 minutes of membrane rupture at the temperature indicated. To achieve 37°C, a dual heater controller (TC-344A, Warner Instrument Corp) was used, with 2 temperature sensors connected and placed in the recording chamber to ensure the on-site temperature.

Data Analysis
Data were analyzed by using Clampfit in pClamp 6.0.4, Microsoft Excel^®, and Microcal Origin^® 4.1. The Chebyshev transform and Simplex least-squares algorithm provided in Clampfit were used for the exponential fitting of the inactivation kinetics. Other nonlinear curve fittings were performed in Origin 4.1 by using the Marquardt-Levenberg least-squares algorithm. The goodness of the fit was evaluated by visual inspection and comparing ² values. The required number of exponential components was judged by F test.

All data presented in this paper were from the control (wild-type) mouse myocytes, except those indicated. Data are expressed as mean±SEM. Student t test was used to compare unpaired data between 2 groups, and 2-tailed P<0.05 was taken to indicate statistical significance.

	Results

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General Properties of Outward Currents of Adult Mouse Myocytes
Depolarization-activated outward currents of adult mouse myocytes were elicited under voltage-clamp conditions by a series of testing pulses from an HP of –50 mV (Figure 1). As described previously,^{8 9 10 11 12 13} the currents rose rapidly to a peak and then decayed. The outward currents of murine cardiocytes inactivated slowly at room temperature and did not reach a steady state after 1-s depolarization (Figure 1A). Longer-pulse protocols (eg, 3 and 10 s) revealed that 3 to 4 s were necessary for the current to reach a steady-state (Figure 1B and 1C). At 37°C, the current decay was accelerated significantly, and, in all cells studied (n=19), an apparent plateau could be reached within 2 to 3 s (Figure 1E).

View larger version (19K): [in this window] [in a new window]   Figure 1. Depolarization-activated outward current in adult mouse ventricular myocytes. A, B, and C show the recordings from a representative cell by test pulses from HP of –50 mV to 60 mV with 10-mV increments for 1, 3, and 10 s, respectively, at room temperature (23°C). D and E, Current traces obtained from a single cell by a series of depolarizations for 1 and 10 s, respectively. In some recordings, a brief (15 to 20 ms) pulse from HP (–50 mV) to –20 mV was used 5 ms before the test pulses to inactivate INa.

All depolarization-activated outward currents were blocked when the K⁺ in the recording pipettes was replaced with equimolar Cs⁺, suggesting that the measured currents are carried primarily or exclusively by K⁺. Moreover, these currents are Ca²⁺-independent, because they were elicited with an intracellular solution containing 5 mmol/L EGTA and an extracellular solution containing 2 mmol/L Co²⁺.

Voltage Dependence and Activation Properties
We have previously shown that, at room temperature, the decay of the outward current elicited by 1-s depolarizing pulses could be well-fitted by a double exponential function with a fast time constant (_fast) of 33.5±2.0 ms at 60 mV,¹¹ close to that of I_to reported in mouse cardiocytes^{9 12} and other species.^{1 2 3 4 5 6 7} In most LQT mouse myocytes, a single exponential was sufficient to describe the current decay with a time constant similar to _fast observed in control cardiocytes.¹¹ The slowly inactivating component of the outward current (I_slow) in LQT myocytes was either "missing" or significantly suppressed. We concluded that I_slow was the target of the transgene, a truncated Kv1.1 channel gene that encodes only the N-terminus and S1-segment polypeptide. Also, these findings enabled us to use a 200-ms depolarizing prepulse (HP, –50 to 40 mV), 5 ms ahead of the test pulses, to separate I_slow from the fast component, I_to. Increasing the recording temperature from 23°C to 37°C markedly speeded up the activation kinetics of the outward currents and the recovery rate of I_to, making a discrimination of capacitance and the ionic current transient very difficult (data not shown). Therefore, we determined the activation properties of I_slow at room temperature to ensure reliable measurements. As shown in Figure 2A, the 100-ms testing pulses after the prepulse evoked a series of delayed rectifier-like currents with slow inactivation. The current-voltage (I-V) relationship (Figure 2B) for the maximum current during 100 ms depolarization illustrates an outward rectification. Apparent activation of this current was first observed at membrane potentials of approximately –20 mV.

View larger version (18K): [in this window] [in a new window]   Figure 2. Activation properties of the slowly inactivating outward current at 23°C. A, Current traces from a representative cell elicited by a series of test pulses from –40 mV to 50 mV by 10 mV increments. To minimize the contamination of Ito, a prepulse of 200 ms from an HP of –50 mV to 40 mV was applied 5 ms before the test pulses. B, Current-voltage (I-V) relationship of Islow. indicates the maximum current; , the current level at the end of 100-ms pulses respectively. The difference between reflects the inactivation during the period. Mean data were obtained from 8 experiments. C, Steady-state activation of Islow. The tail current elicited by a protocol shown in panel A was measured as the difference between the peak and the end current levels during the 100-ms voltage of –20 mV after the test pulses. The currents were normalized to the maximum tail current. Averaged values from 8 observations were fitted by a single Boltzmann distribution (2=0.00099) with a half-activation voltage of –12.1 mV and a slope factor of 8.2 mV. D, Voltage dependence of the activation time constants () of Islow. was obtained by fitting the activating part of the currents by a single exponential function (n=8). Because no apparent current activated at the membrane voltages lower than –30 mV, time constants were plotted as a function of the test potentials within the range from –20 to 50 mV.

To characterize the steady-state activation properties of I_slow, normalized tail currents were obtained and plotted as a function of test potentials (Figure 2C). For each individual cell, a single Boltzmann function was fitted to these normalized currents:

(1)

where V_m is the membrane voltage, V_1/2 is the voltage at half-maximal activation, and S is a slope factor at V_m=V_1/2. The fitting of individual observations (n=8) gave a V_1/2 of –12.5±2.6 mV and a slope of 7.7±1.0 mV.

The time course of activation could be fitted by a single exponential function with time constants ranging from 3.8±0.8 ms at –20 mV to 2.1±0.5 ms at 50 mV. The voltage dependence of the activation time constants is shown in Figure 2D.

Inactivation Properties
The prepulse protocol separating I_slow from I_to was not suitable for studying the steady-state inactivation and recovery kinetics. In these cases, accurate measurement of the amplitude of I_slow was almost impossible because of the overlapping fast component, I_to. To address this problem, we assumed that I_to was fully inactivated after a 200-ms depolarization step at room temperature or after 100 ms at 37°C in accordance with the time constants measured under each condition. The amplitude of I_slow was determined as the difference between the current level at 200 ms depolarization at 23°C (or 100 ms at 37°C) and the current level at the end of the long pulses (5 s). By this method, we obtained a half-inactivation voltage (V_1/2') of -35.1±1.3 mV and a slope factor (S') of -5.4±0.4 mV (n=8) at 37°C (Figure 3). At room temperature, V_1/2' and S' were -37.6±1.8 and -5.8±0.6 mV (n=5), respectively. No significant differences were found between 2 groups (P>0.05), indicating no obvious temperature dependence for the steady-state inactivation of I_slow.

View larger version (11K): [in this window] [in a new window]   Figure 3. Steady-state inactivation of Islow. A, Superimposed current traces evoked by a double-pulse protocol (inset) from a representative cell at 37°C. Pulses (5-s) were used to reach a steady-state inactivation. The voltage protocol was shown in the upper panel. B, Voltage dependence of inactivation. Islow elicited by the second pulse was measured as the difference between the current levels at 200-ms depolarization at 23°C (or 100-ms at 37°C) and at the end of the second pulse. The mean normalized currents to the maximum current were plotted as a function of the test potentials and were well-fitted by a single Boltzmann function (2=0.00024 and 0.0001, respectively, for 37°C and 23°C data) with half-inactivation voltages and slope factors of –35.2 and -5.7 mV at 37°C, and –37.2 and -5.8 mV at 23°C, respectively.

The time course of the outward current inactivation was fitted by a sum of exponentials. With 1-s depolarizing pulses, 2 exponential components could be resolved for all the cells at 23°C (n=14). The resulting time constants (_fast, 30 to 40 ms; _slow, 500 to 600 ms), similar to those reported,^{8 9 11} did not show significant voltage dependence. At 37°C, the current decay with 1-s depolarization seemed to be more complex. Two exponentials best described the time course of current decay in only 6 out of 12 cells. In the other 6 cells (especially those with a large I_to as shown in Figure 4B and 4C), triexponential data fitting was superior to biexponential data fitting. The mean time constants obtained by biexponential and triexponential fittings are plotted against the membrane potentials (Figure 4E), which showed no obvious voltage dependence. Apparently, the fastest time constants (8 to 12 ms) from 3 exponential fittings represented the speeding-up inactivation rate of I_to. Therefore, the shorter time constants in the biexponential fit, compared with those in the triexponential fit, could be explained by a small overlapping fast component (I_to).

View larger version (36K): [in this window] [in a new window]   Figure 4. Inactivation kinetics of Islow. A, Double exponential fit was sufficient to describe the time course of current decay of 1 representative cell (r=0.99962 for the trace at 40 mV). Similar results were observed in 5 other myocytes. Current traces (points) and fitting curves (solid line) were superimposed. B and C, Two exponentials failed well-fitting the current decay (B), whereas 3 exponentials were superior (C) in 1 representative myocytes (F=688.72 for the trace at 40 mV; P<0.001). Similar results were observed in 5 other myocytes. All experiments were performed at 37°C. Currents were elicited by a series of 1-s test pulses from an HP of –60 mV to 50 mV with a 10-mV increment. D and E, Relative amplitudes and the time constants, repectively, of the 2 or 3 components (obtained by bi- or tri-exponential fits, see above) as a function of membrane potential. Open and solid symbols show the mean values obtained by bi- and tri-exponential fits, respectively, from 6 observations.

Ion Selectivity
As mentioned above, all outward currents were blocked when the K⁺ in the recording pipettes was replaced by Cs⁺, suggesting that the measured currents were carried predominantly by K⁺. To examine the K⁺ selectivity of I_slow in greater detail, reversal potentials (E_rev) in response to varying transmembrane K⁺ concentrations ([K⁺]_o) were estimated by using a tail-current protocol shown in Figure 5. Tail currents were measured as the difference between the peak current and the current at the end of the pulse. Because of possible contaminant currents such as I_K1 at relatively negative potentials, it was somewhat difficult to accurately measure the reversal potential of I_slow, especially at high [K⁺]_o when I_K1 was more evident. To minimize the contamination, the reversal potential was determined by extrapolation from the linear part of the relation tail currents to voltages (5-mV increments) close (in most cases, positive) to the reversal potential. In the experiment shown in Figure 5A, I_slow tails reversed between –70 and –75 mV when [K⁺]_o was 5.4 mmol/L. At this [K⁺]_o, E_rev averaged from 7 observations was -71.3±1.2 mV. Figure 5B shows the [K⁺]_o dependence of the reversal potential, which has a linear relationship (r=0.9989; P<0.01) with a slope of 44.3 mV per decade. Mean slopes from 4 individual experiments were 43.4±1.8 mV per decade. These values were lower than an E_rev of –81 mV and a slope of 58.77 mV per decade calculated from the Nernst equation (see figure legend) for a temperature of 23°C. This could be due, at least in part, to the voltage-dependent deactivation process of this current and might also indicate a permeability of ions other than K⁺. A relative permeability of sodium to potassium (P_Na/P_K, P) was then calculated according to the Goldman-Hodgkin-Katz voltage equation¹⁵ :

(2)

where [Na⁺]_o, [Na⁺]_i, [K⁺]_o, and [K⁺]_i were 137.33, 5.0, 5.4, and 130.0 mmol/L, respectively. The resulting P value of 0.019 indicates that I_slow is highly, but maybe not exclusively, K⁺ selective. Chloride ion is unlikely the charge carrier of I_slow, because no significant alterations in the current amplitude, activation/inactivation kinetics, and reversal potential were observed after substituting 100 mmol/L NaCl with equimolar sodium aspartate (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 5. Reversal potential of Islow. A, Tail currents were elicited by membrane hyperpolarizations to potentials between –40 and -85 mV after 100-ms depolarizations to 50 mV from an HP of –50 mV. A prepulse of 200 ms was applied 5 ms before the test depolarizaitons to minimize the contamination of Ito. The voltage protocol was shown in the inset. B, [K+]o dependence of the Erev. Erev were estimated by current-measuring method as described in the text. Averaged data (n=5) were fitted linearly, resulting in a slope of 44.2 mV per decade (r=0.9989, P=0.00107). Dashed line represents a theoretical [K+]o dependence for a pure K+ channel calculated from Nernst Equation, Erev=(-2.303RT/zF)xlog([K+]i/[K+]o), where R is the gas constant, T is the absolute temperature, z is the valence, and F is the Faraday constant.

Recovery From Inactivation
A double-pulse protocol with a variety of intervals was used to study the time-dependent recovery kinetics of I_slow from inactivation (Figure 6). Duration (5-s; or 3-s, for experiments at 37°C) for each pulse was used to fully inactivate this current. I_slow was measured as the difference between the current level at 200- or 100-ms depolarization (for the experiments at 23°C and 37°C, respectively) and the current level at the end of the 5-s (or 3-s, for some experiments as shown in Figure 6A) pulses. The ratios of the currents evoked by the second pulse to the currents by the first pulse were calculated and plotted as a function of the related intervals. At 37°C, 2 exponentials were required to adequately describe the recovery kinetics of I_slow (n=6). Initial recovery was fast with a time constant of 233±34 ms, and 45.5±3.4% amplitude recovered. The remaining part recovered very slowly with a time constant of 3730±702 ms. At room temperature, the recovery process was even slower and incomplete, making curve fitting somewhat difficult. The mean time constants were 830±240 and 8680±2410 ms (n=4) for fast and slow components, respectively. The mean values for both recording conditions shown in Figure 6B were also fitted by biexponential functions. The resulting fast and slow time constants were 250 and 2279 ms at 37°C, respectively and 662 and 6750 ms at 23°C, respectively.

View larger version (27K): [in this window] [in a new window]   Figure 6. Recovery from steady-state inactivation of Islow. A, Superimposed current recordings (37°C) by a series of double pulses from an HP of –50 mV to 50 mV with variable intervals. A 5-s double-pulse protocol was used for the experiments at room temperature. B, Normalized currents as a function of the intervals. Currents were measured as described in Figure 3 and normalized (I2/I1). Averaged values were fitted by a biexponential function with 2 time constants of 250 and 2279 ms at 37°C, and 662 and 6750 ms at 23°C.

Pharmacological Properties
We have shown previously¹¹ that I_slow had a high sensitivity to 4-AP blocking with an IC₅₀ value of 32.1±5.2 µmol/L, whereas I_to was much less sensitive with an 8- to 10-fold higher IC₅₀. Therefore, a low concentration (25 or 50 µmol/L) of 4-AP was employed to block I_slow without significantly affecting I_to. Figure 7B demonstrates that the fast inactivation component remained obvious after the application of 50 µmol/L 4-AP. Subtracting the current after 4-AP application (Figure 7B) from control current (Figure 7A) revealed a rapidly activating and slowly inactivating current that decayed completely after 2 to 3 s (Figure 7C).

View larger version (15K): [in this window] [in a new window]   Figure 7. Pharmacological properties of Islow. A, B, and C, Effects of 4-AP (50 µmol/L) on the outward currents. The 4-AP–sensitive current (C) was obtained by subtraction of the current after the application of 4-AP (B) from that before application (A). D, E, and F, Effects of E-4031 (1 µmol/L) (D), -dendrotoxin (14 nmol/L) (E), and TEA (5 mmol/L) (F) on the outward currents. All experiments were performed at room temperature. and denote before and after drug application.

To further describe the pharmacological properties of I_slow, other probes that are commonly used for identifying the K⁺ channels were also applied to the cells (Figure 7D through 7F). E-4031 at 1 µmol/L (Figure 7D), a specific blocker for I_Kr, and a potent inhibitor for Kv1.2 that encodes a delayed rectifier channel, -dendrotoxin (DTX, 14.3 nmol/L, Figure 7E), did not have a significant effect on the outward current of mouse ventricular myocytes. Another potassium channel blocker, tetraethylammonium (TEA), at 2.5, 5, and 10 mmol/L, had only a slight inhibition on the outward current. The amplitude of the TEA-sensitive current obtained by subtraction method was <10% of total outward current and appeared to have a slow activation and a very slow inactivation (data not shown). In addition, 2 to 10 mmol/L of TEA did not significantly alter APD of the myocytes (data not shown), indicating that the TEA-sensitive current might not play an important role in the formation of the action potential.

Functional Role of I_slow
To evaluate how I_slow functions in the repolarization of adult mouse ventricular myocytes, the effect of 50 µmol/L 4-AP on the action potentials was investigated. Experiments were carried out at 37°C in normal Tyrode's solution under current-clamp mode. Action potentials were elicited by suprathreshold currents injected through the recording electrode at 5 Hz. As demonstrated in Figure 8A, the APD was markedly prolonged after 4-AP application. APD₃₀, APD₅₀, APD₇₀, and APD₉₀ were increased by 66.0±17.5%, 150.0±20.9%, 131.5±18.0%, and 32.3±6.9%, respectively (n=4; P<0.01). In contrast, 4-AP at the same concentration did not exert significant prolongation in LQT ventricular myocytes (Figure 8B).

View larger version (10K): [in this window] [in a new window]   Figure 8. Effect of 4-AP (50 µmol/L) on the action potentials in control (A) and LQT (B) mouse myocytes. Recordings were conducted under current-clamp mode at 37°C. Action potentials were elicited by suprathreshold currents injected through the recording electrode at 5 Hz. indicates recordings before 4-AP applications; , recordings after 4-AP applications; and bars, membrane potentials at 0 mV. Similar results were observed in 3 other observations in each group.

We also applied the recorded action potential waveforms (Figure 9A) to the myocytes under voltage-clamp mode, which elicited a current complex as shown in Figure 9B. 4-AP (25 µmol/L) was then applied, and the 4-AP–sensitive current was obtained by subtraction method. Figure 9D shows that this current reached a peak at approximately 10 mV and vanished at approximately –40 mV, which fits the findings that I_slow activates rapidly and little current is activated at potentials negative to –30 mV. Thus, this current exerts its maximal effect during midrepolarization. Indeed, the LQT myocytes and 4-AP effect (at 25 to 50 µmol/L) showed a larger extent of prolongations in APD₅₀ and APD₇₀.

View larger version (12K): [in this window] [in a new window]   Figure 9. Functional role of Islow in the formation of action potentials. Action potentials were recorded by the method described in the Figure 8 legend. After minimizing the stimulus artifact off-line, the recorded AP (A) was used as a test pulse to stimulate the same cell (1 Hz) under voltage-clamp mode. The elicited currents before and after the application of 4-AP (25 µmol/L) were shown in (B) and (C), respectively. By using digital subtraction, the 4-AP-sensitive current was obtained (D). Similar results were obtained in 3 other experiments.

	Discussion

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I_to and I_slow Are the Two Different Components of the Outward Currents in Adult Mouse Myocytes
We have demonstrated that the outward potassium current in mouse ventricular myocytes has 2 distinct components (I_to and I_slow) that differ in their biophysical and pharmacological properties. The inactivation rate of I_to is approximately 1 order of magnitude faster than that of I_slow. The reactivation of I_to is also much faster than that of I_slow with a time constant of 40 ms at –60 mV (23°C; data not shown). Furthermore, I_slow is significantly more sensitive to 4-AP. The half-inhibition concentration (IC₅₀) of I_to is 10-fold higher than that of I_slow.¹¹ Thus, by applying a prepulse protocol or by using a low concentration of 4-AP, we could distinguish between I_slow and I_to.

The term "transient outward current" has been used previously to describe the total outward current of adult mouse ventricular myocytes. Therefore, on the basis of our findings, the results in some previous experiments could be reinterpreted. Wang and Duff⁹ recently reported a significant developmental increase in the "transient outward current" expression in mouse cardiocytes, which was manifested by a slower inactivation and recovery kinetics and a relatively more positive half-inactivation potential of the "transient outward current" in adult myocytes than those of neonatal cells. It is likely, therefore, that the developmental alterations in the expression level of I_slow underlie these observations. Similarly, a "transient outward current blocker," dibenzylamine, seems to be more potent on I_slow than on I_to,¹⁶ and an anoxia-induced significant reduction in "transient outward current" of adult mouse ventricular myocytes¹⁷ might also be caused mainly by a suppression of I_slow.

We observed that the inactivation of the outward currents was best fitted by 3 exponentials. However, I_slow is unlikely to contribute to the slowest inactivating component on the basis of the following observations: (1) in a few long-pulse experiments (n=4; 23°C) as shown in Figure 7A, we found that the highly 4-AP–sensitive current had only 1 inactivation component with time constants ranging from 300 to 700 ms at different voltages; (2) at 37°C, the current decay (with 1-s depolarization) in LQT myocytes (n=3) could be well-fitted by a 2-exponential function with the fast time constants of 10 ms and slow time constants of 300 ms, close to the fastest and slowest time constants, respectively, found in the controls; and (3) TEA at 10 mmol/L significantly inhibited the sustained current, I_sus. The TEA-sensitive current showed a slow activation and a very slow inactivation with time constants of 1 to 2 s (n=3) at 23°C, indicating that this current may contribute to the slowest inactivating component. The current density of I_sus measured at the end of 5-s depolarization is 6.1±0.4 pA/pF at 50 mV (n=7; 23°C). Taken together, these findings indicate that I_slow has only 1 inactivating component with time constants of 350 ms at 23°C and 100 ms at 37°C with a weak voltage dependence.

I_slow Is Likely Encoded by Kv1.5
Kv1.5 channel has been cloned from mammalian hearts of various species including rat,¹⁸ rabbit,¹⁹ human,^{20 21} and mouse.²² By using a dominant negative approach we demonstrated¹¹ that overexpression of Kv1.1N206, a truncated Kv1.1 cDNA, in the mouse heart resulted in a prolongation of APD and LQT. Previous biochemical studies showed that this transgene retains native Kv1.5-encoded polypeptides in the endoplasmic reticulum and prevents the channel from reaching the plasma membrane.²³ This dominant negative effect is specific against Shaker-like channels (including Kv1.5) but does not affect other voltage-gated channels.²⁴ Electrophysiological studies on the cardiocytes derived from LQT mice revealed that the density of I_slow was significantly reduced as compared with controls. This effect was correlated with a substantial reduction in the steady-state level of Kv1.5 polypeptide expressed in membranes derived from LQT hearts.

A comparison of the biophysical and pharmacological properties of Kv1.5-encoded currents to those of I_slow reveals substantial similarities (Table). Indeed, all Kv1.5-encoded and Kv1.5-like currents are rapidly activating, slowly inactivating, outwardly rectifying, and 4-AP-sensitive. The voltage dependence of channel opening of I_slow and most of the reported currents are similar. The activation time constants of I_slow at very positive membrane potentials are also close to those of the reported currents. At negative potentials, I_slow in mouse myocytes seemed to activate even faster than the other Kv1.5 currents. The sensitivity of I_slow to 4-AP is very close to that of I_Kur and the Kv1.5 channel cloned from human heart, while Kv1.5 cloned from rabbit heart and native Kv1.5-like current in rat atrium are relatively less sensitive to 4-AP. It is unclear whether the difference is caused by different natures of the channels or by the experimental conditions. Furthermore, the inactivation speed of these currents differ significantly from I_slow. Recent work from several laboratories demonstrated that Kvß subunits cloned from the mammalian heart accelerate the inactivation rate of Kv1.5 encoded currents.^{19 28 29 30} The functional differences in Kv1.5 currents expressed in various cell types are probably due to the presence of endogenous Kvß subunits.³⁰ It is therefore likely that the putative ß-subunit(s) expressed in the murine heart is contributing to the faster inactivation rate. Hetero-multimerization of Kv1.5 and other Kv channel (such as Kv1.4) -subunits could also contribute to the relatively fast inactivation of I_slow. London et al¹² reported recently that knock-out of Kv1.4 resulted in a significant decrease in the outward potassium current in mouse cardiocytes. Because the outward currents in their study were elicited by short-pulse (300 to 500 ms) protocols, it is possible that their observation of the reduction in the noninactivating (or slowly inactivating) current could reflect a change in I_slow.

Kv1.4, Kv4.2, and Kv4.3 are known to encode rapidly inactivating currents that resemble the native I_to.³¹ Recent studies^{12 32} suggest that Kv4.2 and Kv4.3, rather than Kv1.4, underlie the rapidly activating and inactivating outward current in mouse hearts. By using a dominant negative approach, Barry et al³² demonstrated that overexpression of a pore mutant of Kv4.2 -subunit (Kv4.2W362F) selectively abolished I_to, leaving a rapidly activating and slowly inactivating current (I_slow) in the mouse ventricular cardiocytes. In contrast, a null mutation of Kv1.4 does not modify the transient outward currents of mouse cardiocytes.¹²

Functional Role of I_slow
By applying a selective concentration of 4-AP that inhibited I_slow by 50% with minimal effect on I_to, we observed a significant prolongation of APD, confirming the important role of I_slow in the repolarization of action potential. Stimulation with action potential waveforms under voltage-clamp mode revealed that I_slow is a major repolarizing current in adult mouse ventricular myocytes, and this current contributes to the early and middle repolarization process. Recent studies demonstrate that in contrast to human where Kv1.5 is primarily expressed in the atrium,^{26 33} the expression level of I_slow and Kv1.5 transcript in murine atria are substantially less compared with those of ventricles.³⁴ In the rat, Kv1.5-like current is mainly expressed in the atrial cells.²⁴ The outward currents of ventricular myocytes contain 2 distinct components³⁵ : the transient outward current and the delayed rectifier (I_K) which is insensitive to 4-AP but blocked by TEA. Thus, the cell-specific expression of Kv1.5 is species specific and is likely regulated by specific sequences located in the promoter.

Potential Limitation and Implications
The presence of multiple overlapping currents in the cardiac myocytes complicates the study of individual K⁺ currents. Therefore, to separate the current of interest from the contaminating currents becomes very important. In this study, we used a prepulse of about 200 ms to inactivate I_to, followed by the test pulses to elicit I_slow. Although the interval (5 ms) was even shorter than those commonly used in the literature, one can never be sure that the contaminating current had been completely removed, especially at body temperature when the recovery speed of I_to was considerably fast. In addition, in some situations where conditioning pulses were not suitable, we measured the difference between the current level at 100-ms depolarization at 37°C (or 200-ms at 23°C) and the current level at the end of the test pulse to represent I_slow. Both this method and the prepulse protocol obviously neglected the decay of I_slow during the initial period, which could amount to 10% to 15% of the total I_slow current according to its time constants. Because only the normalized current ratios were used for further study, the interpretation of our data would not be significantly compromised by this limitation.

All the data presented in this paper were obtained at the presence of 2 mmol/L Co²⁺. We did not choose organic calcium antagonists to block the Ca²⁺ current because many of them have been shown to inhibit Kv1.5-like current.³⁶ However, divalent cations have been reported to cause a shift in voltage dependence of steady-state activation and inactivation of outward currents.³⁷ We also found in our preliminary experiments that 2 mmol/L Co²⁺, in the presence of 5 µmol/L Nifedipine, shifted the steady-state activation and inactivation curves by 15 mV and 7 mV, respectively (n=3), without significantly changing the slope factors and the inactivation kinetics.

Just as the contaminating I_to complicated the analysis of I_slow in this study, caution must be taken when investigating I_to of mouse ventricular myocytes because of the overlap with I_slow. A low concentration of 4-AP can be of great help to minimize it. The protocols commonly used for studying the steady-state inactivation of I_to, that range from 500 to 700 ms, may not be appropriate; a shorter duration (eg, 200 to 250 ms at room temperature) is suggested. This may explain the "incomplete" steady-state inactivation of I_to even at very positive membrane potentials and a very slow recovery time constants reported in previous studies.^{9 10 12} In fact, some of our experiments on I_to showed that a single exponential was sufficient to describe the recovery kinetics of I_to both in control and LQT myocytes when a 200-ms double-pulse protocol was applied. However, a second exponential with a long time constant was required for control (but not LQT) myocytes when a 500-ms protocol was used.

Conclusion
We have characterized the slowly inactivating outward current of adult mouse ventricular myocytes. The biophysical and pharmacological features of this current are similar to those of Kv1.5-encoded channel currents. I_slow plays an important role in the early and middle repolarizing processes of the action potential in adult mouse cardiocytes.

	Acknowledgments

 
This work was supported in part by NHLBI (B.L., G.K.), an AHA Grant-in-Aid (B.L.), and an AHA Established Investigator Award (G.K.). We thank P. Buckett for technical assistance. We wish to thank Dr Shigeru Morishima and Eisai Pharmaceuticals (Japan) for E-4031.

Received March 16, 1998; accepted July 1, 1998.

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