Characterization of a Slowly Inactivating Outward Current in Adult Mouse Ventricular Myocytes
Abstract—We recently have reported that suppression of the slowly inactivating component of the outward current, Islow, 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 Islow 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 Ito, with time constants of ≈100 ms at 37°C. The steady-state inactivation of Islow 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 Islow 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. Islow is highly sensitive to 4-aminopyridine but is insensitive to tetraethylammonium, α-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 Islow are similar to those of Kv1.5-encoded currents.
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 (Ito) 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 Ito (for review, see Giles et al1 ). In some preparations,2 3 4 5 2 components of Ito have been described: Ito1, a Ca2+-dependent and 4-aminopyridine (4-AP)–insensitive outward current, and Ito2, a Ca2+-independent and 4-AP–sensitive outward current. Several recent studies6 7 indicate that the charge carrier of Ito1 is Cl−, rather than K+. In all the preparations, Ito2 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 Ito 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 (Islow). 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
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.
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 MΩ 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 GΩ) 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 (Rs) 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 Rs were within the range of 2 to 10 MΩ, 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 Rs 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 MΩ) were used. These efforts enabled us to minimize the voltage errors (typically not >8 mV) across the electrodes after Rs compensation, and these were not corrected. In all voltage-clamp experiments, Rs compensation was checked regularly to ensure that there were no variations with time. Data were discarded if an increase in Rs 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 GΩ.
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 ICa. 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 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 χ2 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.
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⇓).
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 (τfast) of 33.5±2.0 ms at 60 mV,11 close to that of Ito reported in mouse cardiocytes9 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 τfast observed in control cardiocytes.11 The slowly inactivating component of the outward current (Islow) in LQT myocytes was either “missing” or significantly suppressed. We concluded that Islow 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 Islow from the fast component, Ito. 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 Ito, making a discrimination of capacitance and the ionic current transient very difficult (data not shown). Therefore, we determined the activation properties of Islow 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.
To characterize the steady-state activation properties of Islow, 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: where Vm is the membrane voltage, V1/2 is the voltage at half-maximal activation, and S is a slope factor at Vm=V1/2. The fitting of individual observations (n=8) gave a V1/2 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⇑.
The prepulse protocol separating Islow from Ito was not suitable for studying the steady-state inactivation and recovery kinetics. In these cases, accurate measurement of the amplitude of Islow was almost impossible because of the overlapping fast component, Ito. To address this problem, we assumed that Ito 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 Islow 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 (V1/2′) 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, V1/2′ 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 Islow.
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 (τfast, 30 to 40 ms; τslow, 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 Ito 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 Ito. 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 (Ito).
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 Islow in greater detail, reversal potentials (Erev) 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 IK1 at relatively negative potentials, it was somewhat difficult to accurately measure the reversal potential of Islow, especially at high [K+]o when IK1 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⇓, Islow tails reversed between –70 and –75 mV when [K+]o was 5.4 mmol/L. At this [K+]o, Erev 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 Erev 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 (PNa/PK, P) was then calculated according to the Goldman-Hodgkin-Katz voltage equation15 : 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 Islow is highly, but maybe not exclusively, K+ selective. Chloride ion is unlikely the charge carrier of Islow, 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).
Recovery From Inactivation
A double-pulse protocol with a variety of intervals was used to study the time-dependent recovery kinetics of Islow 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. Islow 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 Islow (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.
We have shown previously11 that Islow had a high sensitivity to 4-AP blocking with an IC50 value of 32.1±5.2 μmol/L, whereas Ito 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 Islow without significantly affecting Ito. 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⇓).
To further describe the pharmacological properties of Islow, 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 IKr, and a potent inhibitor for Kv1.2 that encodes a delayed rectifier channel, α-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 Islow
To evaluate how Islow 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⇓).
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 Islow 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.
Ito and Islow 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 (Ito and Islow) that differ in their biophysical and pharmacological properties. The inactivation rate of Ito is approximately 1 order of magnitude faster than that of Islow. The reactivation of Ito is also much faster than that of Islow with a time constant of 40 ms at –60 mV (23°C; data not shown). Furthermore, Islow is significantly more sensitive to 4-AP. The half-inhibition concentration (IC50) of Ito is 10-fold higher than that of Islow.11 Thus, by applying a prepulse protocol or by using a low concentration of 4-AP, we could distinguish between Islow and Ito.
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 Islow underlie these observations. Similarly, a “transient outward current blocker,” dibenzylamine, seems to be more potent on Islow than on Ito,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 Islow.
We observed that the inactivation of the outward currents was best fitted by 3 exponentials. However, Islow 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, Isus. 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 Isus 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 Islow 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.
Islow 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 Islow 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 Islow 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 Islow and most of the reported currents are similar. The activation time constants of Islow at very positive membrane potentials are also close to those of the reported currents. At negative potentials, Islow in mouse myocytes seemed to activate even faster than the other Kv1.5 currents. The sensitivity of Islow to 4-AP is very close to that of IKur 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 Islow. Recent work from several laboratories demonstrated that Kvβ 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 Kvβ subunits.30 It is therefore likely that the putative β-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) α-subunits could also contribute to the relatively fast inactivation of Islow. 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 Islow.
Kv1.4, Kv4.2, and Kv4.3 are known to encode rapidly inactivating currents that resemble the native Ito.31 Recent studies12 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 α-subunit (Kv4.2W362F) selectively abolished Ito, leaving a rapidly activating and slowly inactivating current (Islow) 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 Islow
By applying a selective concentration of 4-AP that inhibited Islow by 50% with minimal effect on Ito, we observed a significant prolongation of APD, confirming the important role of Islow in the repolarization of action potential. Stimulation with action potential waveforms under voltage-clamp mode revealed that Islow 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 Islow 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 (IK) 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 Ito, followed by the test pulses to elicit Islow. 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 Ito 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 Islow. Both this method and the prepulse protocol obviously neglected the decay of Islow during the initial period, which could amount to 10% to 15% of the total Islow 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 Ito complicated the analysis of Islow in this study, caution must be taken when investigating Ito of mouse ventricular myocytes because of the overlap with Islow. 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 Ito, 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 Ito 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 Ito showed that a single exponential was sufficient to describe the recovery kinetics of Ito 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.
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. Islow plays an important role in the early and middle repolarizing processes of the action potential in adult mouse cardiocytes.
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.
- © 1998 American Heart Association, Inc.
Giles WR, Clark RB, Braun AP. Ca2+-independent transient outward current in mammalian heart. In: Morad M, Ebashi S, Trautwein W, Kurachi Y, eds. Molecular Physiology and Pharmacology of Cardiac Ion Channels and Transporters. Dordresht, Netherlands: Kluwer; 1996:141–168.
Litovsky SH, Antzelevitch C. Transient outward current prominent in canine ventricular epicardium but not endocardium. Circ Res. 1988;62:116–126.
Kenyon JL, Sutko JL. Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers. J Gen Physiol. 1987;89:921–958.
Escande D, Coulombe A, Faivre JF, Deroubaix E, Coraboeuf E. Two types of transient outward currents in adult human atrial cells. Am J Physiol. 1987;252:H142–H148.
Tseng GN, Robinson RB, Hoffman BF. Passive properties and membrane currents of canine ventricular myocytes. J Gen Physiol. 1987;90:671–701.
Zygmunt AC, Gibbons WR. Properties of the calcium-activated chloride current in heart. J Gen Physiol. 1992;99:391–414.
Zygmunt AC. Intracellular calcium activates a chloride current in canine ventricular myocytes. Am J Physiol. 1994;267:H1984–H1995.
Wang L, Duff HJ. Developmental changes in transient outward current in mouse ventricle. Circ Res. 1997;81:120–127.
Zhou J, Hao XM, Wang ZM, Jin MW, Qian JQ, Liu TF. Characterization of outward current in mouse ventricular myocytes. Acta Physiol Sin. 1995;47:535–543.
London B, Jeron A, Zhou J, Buckett P, Han X, Mitchell GF, Koren G. Long QT and ventricular arrhythmias in transgenic mice expressing the N-terminus and first transmembrane segment of a voltage-gated potassium channel. Proc Natl Acad Sci U S A. 1998;95:2926–2931.
Hille B. Ionic Channels of Excitable Membranes. Sunderland, Mass: Sinauer Associates Inc; 1992:1–58.
Koopmann R, Doepner B, Thierfelder S, Hirche H. Dibenzylamine: a new blocker of the transient K+ outward current in isolated ventricular heart cells of mice. Biophys J. 1988;74:A207. Abstract.
Barry DM, Trimmer JS, Merlie JP, Nerbonne JM. Differential expression of voltage-gated K+ channel subunits in adult rat heart: relationship to function K+ channels? Circ Res. 1995;77:361–369.
Tamkun MM, Knoth KM, Walbridge JA, Kroemer H, Roden DM, Glover DM. Molecular cloning and characterization of two voltage-gated K+ channel cDNAs from human ventricle. FASEB J. 1991;5:331–337.
Fedida D, Wible B, Wang Z, Fermini B, Faust F, Nattel S, Brown AM. Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current. Circ Res. 1993;73:210–216.
Attali B, Lesage F, Ziliani P, Guillemare E, Honoré E, Waldmann R, Hugnot JP, Mattéi MG, Lazdunski M, Barhanin J. Multiple mRNA isoforms encoding the mouse cardiac Kv1–5 delayed rectifier K+ channel. J Biol Chem. 1993;268:24283–24289.
Folco E, Mathur R, Mori Y, Buckett P, Koren G. A cellular model for long QT syndrome. J Biol Chem. 1997;272:26505–26510.
Boyle WA, Nerbonne JM. Two functionally distinct 4-aminopyridine-sensitive outward K+ current in adult rat atrial myocytes. J Gen Physiol. 1992;100:1047–1061.
Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes: evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents. Circ Res. 1993;73:1061–1076.
Snyders DJ, Tamkun MM, Bennett PB. A rapidly activating and slowly inactivating potassium channel cloned from human heart: functional analysis after stable mammalian cell culture expression. J Gen Physiol. 1993;101:513–543.
England SK, Uebele VN, Shear H, Kodali J, Bennett PB, Tamkun MM. Characterization of a voltage-gated K+ channel beta subunit expressed in human heart. Proc Natl Acad Sci U S A.. 1995;92:6309–6313.
England SK, Uebele VN, Kodali J, Bennett PB, Tamkun MM. A novel K+ channel beta-subunit (hKv beta 1.3) is produced via alternative mRNA splicing. J Biol Chem. 1995;270:28531–28534.
Uebele VN, England SK, Chaudhary A, Tamkun MM, Snyders DJ. Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv beta 2.1 subunits. J Biol Chem. 1996;271:2406–2412.
Deal KK, England SK, Tamkun MM. Molecular physiology of cardiac potassium channels. Physiol Rev. 1996;76:49–67.
Barry DM, Xu H, Nerbonne JM. Functional knockout of the cardiac transient outward K+ current in mice expressing a dominant negative voltage gated Kv4 α subunit. Biophys J. 1988;74:A27. Abstract.
Feng J, Wible B, Li GR, Wang Z, Nattel S. Antisense oligodeoxynucleotides directed against Kv1.5 mRNA specifically inhibit ultrarapid delayed rectifier K+ current in cultured adult human atrial myocytes. Circ Res. 1997;80:572–579.
Fiset C, Clark RB, Janzen KM, Winkfein R, Giles WR. Molecular identity of K+ currents in adult mouse atrium and ventricle. Biophys J. 1988;74:A207. Abstract.
Apkon M, Nerbonne JM. Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes. J Gen Physiol. 1991;97:973–1011.
Hatem SN, Benardeau A, Rucker-Martin C, Samuel JL, Coraboeuf E, Mercadier JJ. Differential regulation of voltage-activated potassium currents in cultured human atrial myocytes. Am J Physiol. 1996;271:H1609–H1619.
Agus ZS, Dukes ID, Morad M. Divalent cations modulate the transient outward current in rat ventricular myocytes. Am J Physiol. 1991;261:C310–C318.