Characterization of a transient outward K+ current with inward rectification in canine ventricular myocytes

Gui-Rong Li, Haiying Sun, and Stanley Nattel

Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada H1T 1C8

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The threshold potential for the classical depolarization-activated transient outward K+ current and Cl- current is positive to -30 mV. With the whole cell patch technique, a transient outward current was elicited in the presence of 5 mM 4-aminopyridine (4-AP) and 5 µM ryanodine at voltages positive to the K+ equilibrium potential in canine ventricular myocytes. The current was abolished by 200 µM Ba2+ or omission of external K+ (K+o) and showed biexponential inactivation. The current-voltage relation for the peak of the transient outward component showed moderate inward rectification. The transient outward current demonstrated voltage-dependent inactivation (half-inactivation voltage: -43.5 ± 3.2 mV) and rapid, monoexponential recovery from inactivation (time constant: 13.2 ± 2.5 ms). The reversal potential responded to the changes in K+o concentration. Action potential clamp revealed two phases of Ba2+-sensitive current during the action potential, including a large early transient component after the upstroke and a later outward component during phase 3 repolarization. The present study demonstrates that depolarization may elicit a Ba2+- and K+o-sensitive, 4-AP-insensitive, transient outward current with inward rectification in canine ventricular myocytes. The properties of this K+ current suggest that it may carry a significant early outward current upon depolarization that may play a role in determining membrane excitability and action potential morphology.

cardiac electrophysiology; transient outward potassium current; whole cell patch clamp; excitability; barium- and/or potassium-sensitive transient outward peak current

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

DEPOLARIZATION-ACTIVATED outward currents have been distinguished based on differential time- and voltage-dependent properties and pharmacological sensitivity (1), including delayed-rectifier K+ currents (27) and transient outward current (Ito; see Ref. 8). Rapid and slow delayed rectifier K+ currents (IKr and IKs) have been demonstrated in guinea pig (29), canine (36), and human (19) cardiac myocytes. Ultrarapid delayed rectifier K+ current (IKur) has also been described in rat (4), canine (37), and human (34) atrial cells.

Ito has been recognized in cardiac Purkinje fibers since the 1960s (7, 8). Initially believed to be a Cl- current (8), it was subsequently shown in sheep Purkinje fibers to be due predominantly to an increase in K+ conductance (16). The presence of Ito was demonstrated with whole cell voltage-clamp techniques in cardiac cells from a wide range of species, including rat (15), rabbit (10, 11), dog (32, 33), elephant seal (24), ferret (5), and human (2, 11). Kenyon and Gibbons (16) reported that 4-aminopyridine (4-AP) decreased Ito in sheep cardiac Purkinje fibers, and a 4-AP-resistant component of Ito was reduced by Cl- replacement (16). 4-AP has been subsequently used as a selective inhibitor of transient outward K+ current, and 4-AP-sensitive and 4-AP-resistant components of Ito have been reported in sheep Purkinje fibers (6), rabbit ventricular (38) and atrial (39) cells, and canine ventricular (32) and atrial (36) myocytes. The 4-AP-sensitive and 4-AP-resistant components are often termed Ito1 and Ito2, respectively, after Tseng and Hoffman (32). Ito2 has been recognized (38, 39) to be a Ca2+-activated transient outward Cl- current (ICl.Ca).

In canine cardiac myocytes, both delayed-rectifier K+ currents and Ito (Ito1 and Ito2) have been demonstrated to play an important role in myocardial action potential repolarization. We have recently described a 4-AP-resistant Ito with inward rectification in canine ventricular myocytes in a preliminary report (20). The present study was designed to 1) characterize the voltage and time dependence of the current, 2) determine current-voltage (I-V) relation, and 3) assess whether this Ito may be significant during the action potential.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Myocyte isolation. Left ventricular tissues from isolated hearts were obtained via a left thoracotomy after dogs were anesthetized with intravenous pentobarbital sodium (30 mg/kg). All hearts were initially placed in oxygenated Tyrode solution, the left anterior descending coronary artery was cannulated, and ventricular cells were enzymatically isolated with a procedure described previously for human ventricular cell isolation by Li et al. (19). Myocytes were isolated from the digested tissue, placed in a high-K+ storage solution (19), and kept in the medium for at least 1 h before use. A small aliquot of the solution containing the isolated cells was placed in an open perfusion chamber (1 ml) mounted on the stage of an inverted microscope. Experiments were conducted at 36°C. Only quiescent rod-shaped cells showing clear cross-striations were used.

Solutions. Tyrode solution contained (in mM) 136 NaCl, 5.4 KCl, 1.0 MgCl2, 2.0 CaCl2, 0.33 NaH2PO4, 10.0 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), with pH adjusted to 7.4 with NaOH. When Na+ current (INa) was eliminated, NaCl in Tyrode solution was replaced by equimolar choline chloride. The pipette solution contained (in mM) 20 KCl, 110 potassium aspartate, 1.0 MgCl2, 10 HEPES, 5.0 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 GTP, 5 Mg2ATP, and 3 Na2-phosphocreatine, with pH adjusted to 7.2 with KOH. Ryanodine (5 µM) was used to inhibit ICl.Ca (or Ito2), 4-AP (5 mM) was used to block Ito1, and CdCl2 (300 µM) was used to block the Ca2+ current (ICa).

Electrophysiology and data analysis. Membrane currents and/or action potentials were recorded with the tight-seal whole cell patch-clamp techniques and with the use of an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Data were acquired with command pulses that were generated by a 12-bit digital-to-analog converter controlled by pClamp software (Axon Instruments). Recordings were low-pass filtered at 2 kHz and were stored on the hard disk of an IBM-compatible computer.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Depolarization-elicited currents. A: representative tracings of depolarization-activated transient outward currents in the voltage range of -70 to 0 mV (voltage protocol shown in inset of B) in a canine ventricular cell. B: Ba2+ at 200 µM fully inhibited the transient outward current activated by depolarization and decreased the holding current. C: Ba2+-sensitive current obtained by subtracting currents before and after application of Ba2+, showing voltage-dependent inward rectification.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of omission of external K+ (K+o) on depolarization-elicited currents. A: representative tracings of depolarization-activated transient outward currents in the voltage range of -70 to 0 mV (voltage protocol shown in inset of B) in a canine ventricular cell. B: omission of K+o abolished the transient outward current activated by depolarization and decreased the holding current. C: K+o-sensitive current obtained by subtracting currents before and after omission of K+o, showing voltage-dependent inward rectification.

Borosilicate glass (1.0 mm OD) pipettes were prepared with the use of a Brown-Flaming puller (model P87) to produce a tip resistance of 2-3 MOmega when filled with the solution described above. Tip potentials were compensated before the pipette touched the cell. A giga seal was obtained, and the cell membrane was ruptured by gentle suction to establish the whole cell configuration. The series resistance (Rs) was electrically compensated to minimize the duration of the capacitive transient. After compensation, Rs was 2.1 ± 0.4 MOmega . Myocytes were current clamped to record action potentials and/or were voltage clamped to record membrane currents with the use of pClamp6 software. The liquid junctional potential was determined by immersing the pipette into the bath filled with identical pipette solution, and the voltage reading on the amplifier was set to zero by adjusting the offset. The pipette was subsequently placed into the external solution, and the voltage change gives the liquid junction potential. The average junction potential (12 pipettes) was 10.5 ± 0.3 mV, which was not corrected in the experiments.

Nonlinear curve-fitting techniques (Clampfit in pClamp or Sigmaplot; Jandel Scientific, San Rafael, CA) based on the Marquardt procedure were used to fit equations to experimental data. Paired and unpaired Student's t-tests were used as appropriate to evaluate the statistical significance of differences between two group means. Analysis of variance was used for multiple groups. Values of P < 0.05 were considered to indicate significance. Group data are expressed as means ± SE.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ito induced by depolarization in canine ventricular myocytes. Outward currents were elicited by 300-ms depolarizing voltage steps between -70 and 0 mV from a holding potential of -80 mV with 10-mV increments every 10 s. INa was suppressed by replacement of external Na+ with equimolar choline. Ito1 was blocked by 5 mM 4-AP, Ito2 was inhibited with 5 µM ryanodine, and ICa was blocked by 300 µM Cd2+. Figure 1A displays current responses in a representative cell to voltage steps to values between -70 and 0 mV (protocol shown in Fig. 1B, inset). Figure 1B shows that the depolarization-activated current was fully blocked by the addition of 200 µM Ba2+. The Ba2+-sensitive current is shown in Fig. 1C. Both original and Ba2+-sensitive currents show a transient outward behavior and voltage-dependent inward rectification. The Ba2+-sensitive Ito was carried by neither Ito1 (since it was 4-AP insensitive) nor by Ito2 (seen in the presence of ryanodine and absence of ICa).

Figure 2 shows the effect of the omission of external K+ (K+o) on depolarization-induced Ito. Figure 2A displays currents elicited in a representative cell by the voltage-clamp protocol shown in Fig. 2B, inset. Figure 2B shows that the omission of K+o abolished the depolarization-elicited outward current. K+o omission-sensitive (K+o-sensitive) current is displayed in Fig. 2C. As was the case for the Ba2+-sensitive current, the K+o-sensitive current also shows voltage-dependent inward rectification. In subsequent experiments, the current properties were analyzed by the use of currents sensitive to Ba2+ and/or K+o omission.

Voltage dependence of Ito sensitive to Ba2+ or K +o omission. To study voltage dependence of the depolarization-activated Ito, myocytes were depolarized to more positive potentials using the same voltage-clamp protocol as in Fig. 1B. Because the activation of the Ito was too fast to separate from membrane capacitance at more positive potentials, Ba2+-sensitive and/or K+osensitive current was used to determine the I-V relation for membrane currents. Figure 3 shows Ba2+-sensitive currents from a representative cell at depolarization voltages between -70 and +40 mV from a holding potential of -80 mV. Figure 3A displays current recordings at potentials between -70 and -40 mV. The Ito was seen at -70 mV in this cell, just positive to the resting potential of -71 mV, as indicated by the step from the holding current. The Ito was augmented as the depolarization potential was made more positive. The current showed voltage-dependent inward rectification in the voltage range -30 to 0 mV (Fig. 3B) and reached a steady-state level at potentials between +10 and +40 mV (Fig. 3C).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   Voltage dependence of depolarization-activated Ba2+-sensitive current. Ba2+-sensitive currents were obtained by subtracting currents before and after the addition of 200 µM Ba2+ as in Fig. 1C. Current tracings are shown at potentials between -70 and -40 mV (A), between -30 and 0 mV (B), and between +10 and +40 mV (C).

Figure 4A illustrates schematically the method of current measurement in a Ba2+-sensitive current recording and indicates the measurement for the total current (Itotal), the time-dependent current peak (Ipeak), the steady-state current (Iss), and the holding current (Ih). For analyses of average currents, the measured currents were normalized to cell capacitance to evaluate current density and thereby control for variations in cell size.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4.   Current-voltage (I-V) relations of depolarization-elicited currents. A: schematic showing current measurement in a Ba2+-sensitive current tracing recorded with 300-ms step to -30 mV from a holding potential of -80 mV. Itotal, total current; Ipeak, time-dependent peak current; Iss, steady-state current; Ih, holding current. B: I-V relations of Ba2+-sensitive component (n = 20) for Itotal (open circle ), Ipeak (bullet ), Iss (triangle ), and Ih (down-triangle), extrapolated to the I-V curves. C: I-V relations of K+o-sensitive component (n = 15). Ba2+- or K+o-sensitive currents were obtained as in Figs. 1C or 2C. Data are means ± SE. TP, test potentials.

Figure 4B shows mean density-voltage relations for Ba2+-sensitive currents (n = 20 cells). Itotal and Ipeak density increased to reach a maximum at about -10 and +10 mV, respectively, and then showed inward rectification. The I-V curve for Iss displayed strong inward rectification positive to -50 mV, similar to that of inwardly rectifying K+ current (IK1), suggesting that Iss may be IK1 or a component of IK1. Extrapolating the I-V curves to the voltage axis, the reversal potentials (VRev) for Itotal, Ipeak, and Iss were between -70 and -74 mV. Correcting for the average 10.5-mV liquid junction potential, the VRev would be -80.5 to -84.5 mV, very close to the calculated K+ equilibrium potential (EK, -84 mV). This suggests that the depolarization-induced Ba2+-sensitive current is carried by K+. Figure 4C displays mean density-voltage relations of K+o-sensitive currents obtained as in Fig. 2C. The I-V relations obtained with K+o-sensitive currents (n = 15 cells) were similar to those obtained with Ba2+-sensitive currents, suggesting that the concept that Ba2+-sensitive or K+o-sensitive currents are the same.

Ko dependence and Ba2+ concentration-dependent inhibition of the depolarization-induced current. The K+ selectivity of Ba2+-sensitive Ito was further assessed by evaluating the response of the VRev to various K+o concentrations ([K+]o). The VRev of the Ito was determined by extrapolating the time-dependent peak current to Ih. A 10-fold change in K+o led to a 52 ± 2 mV/decade (n = 6) shift in the VRev value (Fig. 5A), in agreement with the prediction of the Nernst equation (60 mV/decade). To determine whether Ipeak and Iss have similar sensitivity to Ba2+, the 50% inhibitory concentration (IC50) of Ba2+ on Ipeak and Iss was assessed in six cells using 300-ms steps to -20 mV from a holding potential of -80 mV. IC50 was 5.0 ± 0.2 µM for Ipeak and 4.9 ± 0.2 µM for Iss (P = not significant; Fig. 5B), indicating identical and very high Ba2+ sensitivity.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   A: relation between Ipeak reversal potential (RP) and extracellular K+ concentration ([K+]o; means ± SE, n = 6 at each [K+]o). Best-fit regression line (shown) had a slope of 51.5 mV/decade change in [K+]o. B: concentration-dependent effect of Ba2+ on depolarization-elicited currents. Concentration-response curves shown are fitted by the equation E = Emax/[1 + (K/C)n], where E is the effect at a concentration C, Emax is the maximum effect, K is the concentration for half-maximum action, and n is the Hill coefficient. No significant difference in the 50% inhibitory concentration of Ba2+ on Ipeak and Iss was observed with the use of 300-ms steps to -20 mV from a holding potential of -80 mV. Data are means ± SE (n = 6). [Ba2+], Ba2+ concentration.

Figure 6 shows the voltage dependence of Ipeak (A) and Iss (B) in the presence of different [K+]o (2.7, 5.4, and 10.8 mM). The I-V curves for Ipeak and Iss shifted to the right with increased EK. The VRev of Iss shifted with [K+]o like that of Ipeak; however, the maximum amplitude of Iss increased as [K+]o increased. This behavior suggests increased conductance at higher [K+]o, behavior typical of IK1.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of the change of [K+]o (2.7, 5.4, and 10.8 mM) on the voltage dependence of K+o-sensitive currents. A: voltage dependence of Ipeak. I-V relation curves shifted to the right along with the change of the K+ equilibrium potential (EK). B: voltage dependence of Iss. I-V relation curves shifted to the right, and conductance of the current increased along with the alteration of EK. Data are means ± SE; n = 7, 10, and 7, respectively, for 2.7, 5.4, and 10.8 mM K+o.

The results indicate that the addition of Ba2+ and the omission of K+o strongly suppress the depolarization-elicited Ito. Clearly, Ba2+- and/or K+o-sensitive current is not classical 4-AP-sensitive Ito1, because 1) the threshold voltage of activation for Ito1 is -30 to -20 mV in canine ventricular myocytes (21), and 2) experiments for the determination of the current are conducted in the presence of 5 mM 4-AP, which fully blocks Ito1. We will refer to the Ba2+- and/or K+o-sensitive transient outward peak current (Ipeak) with inward rectification as Ito.ir.

Kinetics of time-dependent Ito.ir. The time dependence of Ito.ir was assessed as illustrated in Fig. 7A, which shows representative 200 µM Ba2+-sensitive current obtained during a 300-ms voltage step from -80 to -30 mV. The raw data were best fitted by a biexponential equation with time constants shown. Mean data for inactivation time constants in 12 cells are shown in Fig. 7B. At all voltages, inactivation of the current was well fitted by a biexponential relation and poorly fitted by a monoexponential function. The fast inactivation time constant showed significant voltage dependence (P < 0.01), indicating that rapid inactivation is faster when the test potential is positive to -10 mV. However, the slow inactivation time constant showed no significant voltage dependence (P > 0.05).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Time-dependent inactivation of Ba2+- and/or K+o-sensitive transient outward peak current (Ito.ir). A: inactivation of Ba2+-sensitive Ito.ir on 300-ms depolarization from -80 to -30 mV was fitted by a biexponential function (curve shown as solid line, points are raw data). B: rapid and slow time constants (tau 1 and tau 2, respectively) of Ito.ir inactivation obtained with the approach illustrated in A (n = 12). Ba2+-sensitive current was obtained as in Fig. 1C. Data are means ± SE.

Voltage dependence of Ito.ir inactivation was determined as illustrated in Fig. 8A. Prepulses of 300-ms duration were applied to conditioning potentials between -120 and 0 mV, and then currents were recorded during 300-ms test pulses to +10 mV before and after 200 µM Ba2+. The inactivation variable of Ba2+-sensitive Ito was determined as Ito.ir at a given prepulse potential divided by the maximum Ito.ir in the absence of a prepulse. Figure 8B shows mean results obtained from analysis of voltage-dependent inactivation. Inactivation reached a maximum at 0 mV and was incomplete. Mean data are represented with filled circles, and the curve is the best-fit Boltzmann distribution. The half-inactivation voltage (V0.5) averaged -43.5 ± 3.2 mV (n = 11), whereas the slope factor averaged 12.6 ± 0.7 mV.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 8.   Voltage-dependent inactivation of Ito.ir. A: protocol and representative Ba2+-sensitive current tracings used to assess voltage-dependent inactivation of Ito.ir. B: mean voltage-dependent inactivation relation for Ito.ir. Inactivation was assessed with the protocol shown in A. Data were fit to Boltzmann relation of I/Imax = [1 + exp(V-V0.5)/K)]-1, where I/Imax is the inactivation variable of Ito.ir, V is the membrane potential, V0.5 is the membrane potential for half-maximal inactivation, and K is a slope factor. The Ba2+-sensitive current was obtained as in Fig. 1C. Data are means ± SE. CP, conditioning potentials.

The time dependence of reactivation of Ba2+-sensitive Ito.ir from inactivation was studied with the paired-pulse protocol illustrated in Fig. 9A. Identical 300-ms pulses (P1 and P2) from the holding potential (-80 mV) to -20 mV were delivered every 10 s, with varying P1-P2 intervals. The current during P2 (I2) relative to the current during P1 (I1) was determined as a function of the P1-P2 recovery interval. The curve in Fig. 9B shows a nonlinear exponential curve fit to mean data from 10 cells. The reactivation curve of Ito.ir was well fitted in individual experiments by monoexponential functions with time constants averaging 13.2 ± 2.5 ms.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 9.   Time-dependent reactivation of Ito.ir. A: representative Ba2+-sensitive current tracings (obtained as in Fig. 1C) for assessing reactivation of Ito.ir from inactivation obtained with 2 identical pulses shown in inset (300 ms from the holding potential -80 to -20 mV), P1 and P2, with varying P1-P2 interval. B: reactivation curve for Ito.ir was best fitted by a monoexponential function with inactivation time constant of 12.5 ms (n = 10). Data are means ± SE. I1, current during P1; I2, current during P2; Delta t, change in time of P1 and P2 interval.

Effects of intracellular Mg2+ and spermine on Ito.ir. Intracellular Mg2+ (Mg2+i) and polyamines, such as spermine, have been reported to be related to inward rectification property of IK1 and/or cloned channels with inward rectification (25). We therefore omitted Mg2+i or included 5 µM spermine in the pipette solution to observe whether Ito.ir was changed after cell dialysis for 10 min. The omission of Mg2+i did not induce a significant change in Ito.ir (Fig. 10). Similarly, spermine inclusion did not significantly change the current amplitude or I-V relation of Ito.ir either. At a voltage step to -40 mV from -80 mV, Ito.ir was 3.9 ± 0.8 (n = 8), 3.8 ± 0.7 (n = 4), and 4.1 ± 1.1 (n = 4) pA/pF, respectively, under control, Mg2+i-free, and spermine-inclusion conditions.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 10.   Effect of omitting intracellular Mg2+ (Mg2+i) on Ito.ir. A: current recordings in a control cell. B: current recordings in another cell dialyzed with a Mg2+-free pipette solution for 10 min. Ba2+-sensitive Ito.ir was obtained as in Fig. 1C.

Comparison between 4-AP-sensitive Ito1 and K+osensitive Ito.ir. All of the above studies of Ba2+- or K+o-sensitive Ito.ir were performed in the presence of 5 mM 4-AP, excluding the participation of classical 4-AP-sensitive Ito1. It was of interest to compare directly some of the properties of 4-AP-sensitive Ito1 with Ito.ir sensitive to K+o omission. The two components were separated by the application of 5 mM 4-AP and subsequent omission of K+o in the presence of 5 µM ryanodine. Membrane currents were elicited by 300-ms voltage steps to between -70 and +40 mV from a holding potential of -80 mV, with 10-mV increments applied every 10 s. Figure 11, A-E, displays control currents (A), currents after 4-AP application (B), 4-AP-sensitive Ito1 (C), currents after K+o omission (D), and K+o omission-sensitive currents (E) in a representative cell. 4-AP-sensitive Ito1 (C) is clearly different in time course and voltage dependence from the K+o-sensitive component (E). The I-V relations of the time-dependent peak current for 4-AP-sensitive and K+o-sensitive components are shown in Fig. 11F. 4-AP-sensitive Ito1 shows a linear (ohmic) I-V relation and an activation threshold potential at about -20 mV, whereas the K+o-sensitive Ito.ir shows an I-V relation with inward rectification and a threshold voltage at about -70 mV.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 11.   Difference between 4-aminopyridine (4-AP)-sensitive Ito1 and 4-AP-insensitive, K+o-sensitive Ito.ir. A: membrane current in control. B: recordings after the application of 5 mM 4-AP for 10 min. C: 4-AP-sensitive current obtained by subtracting currents before and after the application of 4-AP. D: recordings after the omission of K+o in the same cell. E: K+o-sensitive current obtained by subtracting currents before and after the omission of K+o. F: I-V relation curves of 4-AP-sensitive Ito1 and K+o-sensitive Ito.ir in the same 5 cells; data are means ± SE. Experiments were conducted with external choline solution in the presence of 300 µM Cd2+ and 5 µM ryanodine.

Figure 12 displays I-V relations of 4-AP-sensitive Ito1 and K+o-sensitive Ito.ir at holding potentials of -80, -70, and -60 mV. Ito1 was not affected by the alteration of holding potentials (Fig. 12A), indicating that Ito1 is not inactivated over the holding potential range tested, consistent with previous observation (21). However, K+o-sensitive Ito.ir was significantly decreased by changing holding potentials from -80 to -70 and -60 mV (Fig. 12B; n = 6, P < 0.01), suggesting that this current, unlike Ito1, may undergo inactivation at holding potentials between -80 and -60 mV. These findings are consistent with the voltage dependence of Ito.ir inactivation shown in Fig. 8 and point out the differences in basic biophysical properties between 4-AP-sensitive Ito1 and Ba2+- or K+o-sensitive Ito.ir.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 12.   Effects of the holding potential (Vh) on 4-AP-sensitive Ito1 and K+o-sensitive Ito.ir (obtained as in Fig. 11) in the same cells. A: I-V relation curves of 4-AP-sensitive Ito1 at holding potentials of -80, -70, and -60 mV. No significant difference in Ito1 was observed in the holding potentials tested. B: I-V relation curves of K+o-sensitive Ito.ir at holding potentials of -80, -70, and -60 mV. Ito.ir significantly decreased as the holding potential changed to -70 and -60 mV from -80 mV. ** P < 0.01 compared with the previous holding potential in all test potentials. Data are means ± SE; n = 6.

Contribution of Ito.ir to the action potential. We evaluated the possible contribution of Ito.ir to the action potential with the use of the action potential clamp technique. The action potential was first recorded in current-clamp mode in normal Tyrode solution and was then loaded as a voltage-clamp waveform to record the membrane currents in choline solution in the presence of 5 mM 4-AP, 5 µM ryanodine, and 300 µM Cd2+. The membrane current during the action potential was obtained by subtracting membrane currents before and after the addition of 200 µM Ba2+. Similarly, a ramp protocol was used in the same cell.

Figure 13 shows representative recordings from a canine ventricular cell. Figure 13A shows the action potential waveform, and Fig. 13B displays current tracings recorded with action potential clamp in the absence (control) and presence of 200 µM Ba2+. Figure 13C displays Ba2+-sensitive current obtained by subtracting the current in the presence of Ba2+ from the control current. Two components of Ba2+-sensitive currents were revealed during the action potential, one transient outward component evident immediately after depolarization and another component during phase 3 repolarization corresponding to classical IK1. Little current was present during the plateau of the action potential. In the same cell, Ba2+-sensitive current was assessed by a ramp protocol (Fig. 13D). Membrane currents activated by the ramp protocol are shown in Fig. 13E in the absence and presence of Ba2+. Similarly, two components of Ba2+-sensitive currents were also elicited by the ramp protocol (Fig. 13F). Similar results were obtained with both of the protocols in a total of six cells.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 13.   Contribution of Ito.ir to action potential. A: action potential from a canine ventricular myocyte was used to clamp the same cell. B: membrane currents activated by the action potential waveform before (control) and after exposure to 200 µM Ba2+. C: Ba2+-sensitive current during the action potential, obtained by subtracting membrane currents before and after the addition of Ba2+. D: ramp protocol used to clamp the same cell. E: membrane currents were recorded by the ramp protocol before (control) and after the addition of 200 µM Ba2+. F: Ba2+-sensitive current during the ramp protocol obtained by subtracting membrane currents before and after the addition of Ba2+.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we have demonstrated that 1) depolarization elicits a 4-AP-resistant Ito at physiological temperature in canine ventricular myocytes under conditions that prevent intracellular Ca2+ transient, 2) the depolarization-induced current inactivates quickly, 3) the current is fully suppressed by the application of Ba2+ (200 µM) or the removal of K+o, and 4) the VRev of the Ba2+- and K+o-sensitive current responds to changes in [K+]o. This novel transient outward component (Ito.ir) shares some features (sensitivity to Ba2+ addition and K+o removal, inward rectification) with IK1. Some properties of Ito.ir are close to those of outward current carried by the recently cloned human TWIK-1 channel (18).

Comparison with previously reported Ito. Two types of Ito have been reported in mammalian cardiac myocytes (32, 38, 39). One is 4-AP-sensitive (Ito1), and the another is a Ca2+-activated Cl- current (Ito2). The threshold potential for Ito1 activation is -30 to -20 mV, and the I-V relation curve is linear (Fig. 10F; see Ref. 21). Ito2 is activated at -30 mV, corresponding to the activation of ICa and intracellular Ca2+ transient, and the I-V relation curve is bell shaped (38, 39). Ito.ir is apparent at -70 to -60 mV, just positive to the current reversal potential (e.g., EK), and the I-V relation curve is neither linear nor bell shaped. Ito.ir is present under conditions (5 mM 4-AP, 5 µM ryanodine, and 300 µM Cd2+) that fully suppress Ito1 and Ito2 and has biophysical properties that are clearly different from the two latter, well-described currents.

Martin et al. (22) reported a 3,4-diaminopyridine-insensitive, Ca2+-independent transient outward K+ current in feline cardiac ventricular myocytes (22). The current was not activated upon initial whole cell patch formation, but activation depended on patch duration, and it was therefore named patch duration-dependent K+ current [IK(PDD)]. IK(PDD) shares some properties with Ito.ir described in the present paper, such as 4-AP insensitivity, Ba2+ sensitivity, time-dependent recovery, and voltage-dependent inactivation. However, the Ito.ir that we studied was developed upon membrane rupture, and its I-V relation (with inward rectification) is clearly different from that of IK(PDD), which is linear in the voltage range of -40 to +60 mV.

Comparison with previously reported outward currents with inward rectification. IKr has been demonstrated to show inward rectification and to have a bell-shaped I-V relation, but the current is not transient (29). We were able to detect a large Ito.ir in the presence of 5 µM E-4031 (n = 5, data not shown), excluding the participation of IKr.

It is known that IK1 can exhibit time and voltage dependence (17); however, time- and voltage-dependent phenomena are well recognized only at membrane potentials negative to the normal resting potential (12). The I-V relation of depolarization-induced instantaneous IK1 was reported to be linear under high K+o (14-40 mM); however, the monoexponential inactivation time constant of instantaneous IK1 is in the range of 1.1 (14) to 7.7 (17) ms. The inward rectification of IK1 has been thought to be related to the action of Mg2+i (17) and polyamines (25) on this channel and/or intrinsic, voltage-dependent gating or closing of the IK1 channel (17). Therefore, the IK1 channel has been considered to act as a diode (23) active only on hyperpolarization of the membrane (26). The single channel conductance is nearly ohmic in the inward direction (17, 23, 27), and it is believed that very little or no current passes through the channel in the outward direction under physiological conditions (23, 33). Shimoni et al. (30) reported that IK1 was inactivated during the upstroke and plateau phases of the action potential and is consequently available for repolarization only during phase 3.

The Ito.ir that we studied shares some features (sensitivity to Ba2+ addition and K+o removal, inward rectification) with IK1 but was not affected by omitting Mg2+i or including spermine in the pipette solution. This may be related to the possibility that submillimolar endogenous polyamines mask the action of Mg2+i removal or micromolar spermine inclusion. Although studies from Tourneur et al. (31) and Ibarra et al. (13) suggested that IK1 may also play an active role during action potential depolarization, the kinetics and I-V relation of depolarization-activated outward IK1 have not been directly analyzed experimentally. Ito.ir could be carried by IK1, a component thereof, or by a previously unidentified channel that shares many feature with IK1. Ito.ir shows features that are consistent with the idea of an intrinsic voltage-dependent inactivation mechanism (25). The different response of Ipeak (Ito.ir) and Iss to changes in [K+]o (Fig. 6) suggests that they may not be carried by the same mechanism, with the Iss responding more like classical IK1. We have observed Ito.ir in ventricular myocytes from guinea pig, rabbit, and human hearts (unpublished data), indicating widespread expression in mammalian hearts.

Potential significance of our observation. Cell excitability has generally been associated with the ability of inward currents to generate an action potential upstroke. More recent studies suggest that IK1 may also play a role in the excitability of cardiac cells (13) by stabilizing the resting potential (25). Because significant outward currents carried by Ito.ir can be elicited by depolarization over a time course comparable to INa, this outward component may play a role in cell excitability, especially when INa is reduced, as in myocardial ischemia. Recent work suggests that voltage-dependent changes in maximal velocity with increased [K+]o are poorly explained by changes in INa (35). This discrepancy may be due to a participation of Ito.ir in determining maximal velocity, which becomes particularly important when INa is reduced.

    ACKNOWLEDGEMENTS

This work was supported by the Heart and Stroke Foundation of Québec, and Fonds de la Recherche en Santé du Québec (FRSQ).

    FOOTNOTES

G.-R. Li is a research scholar of FRSQ.

Address for reprint requests: G.-R. Li, Montreal Heart Institute, 5000 Belanger St. East, Montreal, Quebec, Canada H1T 1C8.

Received 6 August 1997; accepted in final form 14 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Barry, D. M., and J. M. Nerbonne. Myocardial potassium channels: electrophysiological and molecular diversity. Annu. Rev. Physiol. 58: 363-394, 1996[Medline].

2.   Beuckelmann, D. J., M. Näbauer, and E. Erdmann. Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ. Res. 73: 379-385, 1993[Abstract].

3.   Boyett, M. R. Effect of rate-dependent changes in the transient outward current on the action potential in sheep Purkinje fibres. J. Physiol. (Lond.) 319: 23-41, 1981[Abstract].

4.   Boyle, W. A., and J. M Nerbonne. A novel type of depolarization-activated K+ current in isolated adult rat atrial myocytes. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H1236-H1247, 1991[Abstract/Free Full Text].

5.   Campbell, D. L., Y. Qu, R. L. Rasmusson, and H. C. Strauss. The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use-dependent block by 4-aminopyridine. J. Gen. Physiol. 101: 603-626, 1993[Abstract].

6.   Coraboeuf, E., and E. Carmeliet. Existence of two transient outward currents in sheep cardiac Purkinje fibers. Pflügers Arch. 392: 352-359, 1982[Medline].

7.   Deck, K., and W. Trautwein. Ionic currents in cardiac excitation. Pflügers Arch. 280: 63-80, 1964.

8.   Dudel, J., K. Peper, R. Rüdel, and W. Trautwein. The dynamic chloride component of membrane current in Purkinje fibers. Pflügers Arch. 295: 197-212, 1967.

9.   Dukes, I. D., and M. Morad. The transient K+ current in rat ventricular myocytes: evaluation of its Ca2+ and Na+ dependence. J. Physiol. (Lond.) 435: 305-420, 1990.

10.   Fedida, D., and W. R. Giles. Regional variations in action potentials and transient outward current in myocytes isolated from rabbit left ventricle. J. Physiol. (Lond.) 442: 191-209, 1991[Abstract].

11.   Fermini, B., Z. Wang, D. Duan, and S. Nattel. Differences in rate dependence of transient outward current in rabbit and human atrium. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1747-H1754, 1992[Abstract/Free Full Text].

12.   Harvey, R. D., and R. E. Ten Eick. Characterization of the inward-rectifying potassium current in cat ventricular myocytes. J. Gen. Physiol. 91: 593-615, 1988[Abstract].

13.   Ibarra, J., G. E. Morley, G. E., and M. Delmar. Dynamics of the inward rectifier K+ current during the action potential of guinea pig ventricular myocytes. Biophys. J. 60: 1534-1539, 1991[Abstract].

14.   Ishihara, K., T. Mitsuiye, A. Norma, and M. Takano. The Mg2+ block and intrinsic gating of underlying inward rectification of the K+ current in guinea pig cardiac myocytes. J. Physiol. (Lond.) 419: 297-320, 1989[Abstract].

15.   Josephson, I. R., J. Sanchez-Chapula, and A. M. Brown. Early outward current in rat single ventricular myocytes. Circ. Res. 54: 157-162, 1984[Abstract].

16.   Kenyon, J. L., and W. R. Gibbons. 4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibres. J. Gen. Physiol. 73: 139-157, 1979[Abstract].

17.   Kurachi, Y. Voltage-dependent activation of the inward-rectifier potassium channel in the ventricular cell membrane of guinea-pig heart. J. Physiol. (Lond.) 366: 365-385, 1985[Abstract].

18.   Lesage, L., E. Guillemare, M. Fink, F. Duprat, M. Lazdunski, G. Romey, and J. Barhanin. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. EMBO J. 15: 1004-1011, 1996[Abstract].

19.   Li, G. R., J. Feng, L. Yue, M. Carrier, and S. Nattel. Evidence for two components of delayed rectifier K+ currents in human ventricular myocytes. Circ. Res. 75: 123-136, 1996[Abstract].

20.   Li, G. R., H. Sun, and S. Nattel. The inward rectifier contributes to 4-aminopyridine resistant transient outward current during the canine ventricular action potential (Abstract). J. Am. Coll. Cardiol. 29: 513A-514A, 1997.

21.   Liu, D. W., A. Gintant, and C. Antzelevitch. Ionic bases of electrophysiological distinction in canine ventricular myocytes. Circ. Res. 72: 672-702, 1993.

22.   Martin, R. L., P. L. Barrington, and R. E. Ten Eick. A 3,4-diaminopyridine-insensitive, Ca2+-independent transient outward K+ current in cardiac ventricular myocytes. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H1286-H1299, 1994[Abstract/Free Full Text].

23.   Matsuda, H., A. Saigusa, and H. Irisawa. Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+. Nature 325: 156-159, 1987[Medline].

24.   Maylie, J., and M. Morad. A transient outward current related to calcium release and development of tension in elephant seal atrial fibres. J. Physiol. (Lond.) 357: 267-292, 1984[Abstract].

25.   Nichols, C. G., E. N. Makina, W. L. Pearson, Q. Sha, and A. N. Lopatin. Inward rectification and implications for cardiac excitability. Circ Res. 78: 1-7, 1996[Abstract/Free Full Text].

26.   Noble, D. The surprising heart: a review of recent progress in cardiac electrophysiology. J. Physiol. (Lond.) 353: 1-50, 1984[Medline].

27.   Noble, D., and R. W. Tsien. Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres. J. Physiol. (Lond.) 200: 205-231, 1969[Medline].

28.   Sakmann, B., and G. Trube. Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart. J. Physiol. (Lond.) 347: 641-657, 1984[Abstract].

29.   Sanguinetti, M. C., and N. K. Jurkiewicz. Two components of cardiac delayed rectifier K+ current. J. Gen. Physiol. 96: 195-215, 1990[Abstract].

30.   Shimoni, Y., R. B. Clark, and W. R. Giles. Role of an inwardly rectifying potassium current in rabbit ventricular action potential. J. Physiol. (Lond.) 448: 709-727, 1992[Abstract].

31.   Tourneur, Y., R. Mitra, M. Morad, and O. Rougier. Activation properties of the inward-rectifying potassium channel on mammalian heart cells. J. Membr. Biol. 97: 127-135, 1987[Medline].

32.   Tseng, G. N., and B. F. Hoffman. Two components of transient outward current in canine ventricular myocytes. Circ. Res. 64: 633-647, 1989[Abstract].

33.   Vandenberg, C. G. Cardiac inward rectifier potassium channel. In: Ion Channels in the Cardiovascular System: Function and Dysfunction, edited by P. M. Spooner, A. M. Brown, W. A. Catterall, G. J. Kaczorowski, and H. C. Strauss. Mt. Kisco: NY: Futura, 1994, p. 145-167.

34.   Wang, Z., B. Fermini, and S. Nattel. Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned currents. Circ. Res. 73: 276-285, 1993[Abstract].

35.   Whalley, D. W., D. J. Wendt, C. F. Starmer, Y. Rudy, and A. O. Grant. Voltage-independent effect of extracellular K+ on the Na+ current and phase 0 of the action potential in isolated cardiac myocytes. Circ. Res. 75: 491-502, 1994[Abstract].

36.   Yue, L., J. Feng, G. R. Li, and S. Nattel. Transient outward and delayed rectifier currents in canine atrium: properties and role of isolation methods. Am. J. Physol. 270 (Heart Circ. Physiol. 39): H2157-H2168, 1996[Abstract/Free Full Text].

37.   Yue, L., J. Feng, G. R. Li, and S. Nattel. Characterization of an ultrarapid delayed rectifier potassium channel involved in canine atrial repolarization. J. Physiol. (Lond.) 496: 647-662, 1996[Abstract].

38.   Zygmunt, A. C., and W. R. Gibbons. Calcium-activated chloride current in rabbit ventricular myocytes. Circ. Res. 68: 424-437, 1991[Abstract].

39.   Zygmunt, A. C., and W. R. Gibbons. Properties of the calcium-activated chloride current in heart. J. Gen. Physiol. 99: 424-437, 1992.


AJP Cell Physiol 274(3):C577-C585
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society