INTRODUCTION

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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 (I_to; see Ref. 8). Rapid and slow delayed rectifier K⁺ currents (I_Kr and I_Ks) have been demonstrated in guinea pig (29), canine (36), and human (19) cardiac myocytes. Ultrarapid delayed rectifier K⁺ current (I_Kur) has also been described in rat (4), canine (37), and human (34) atrial cells.

I_to 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 I_to 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 I_to in sheep cardiac Purkinje fibers, and a 4-AP-resistant component of I_to 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 I_to 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 I_to1 and I_to2, respectively, after Tseng and Hoffman (32). I_to2 has been recognized (38, 39) to be a Ca²⁺-activated transient outward Cl current (I_Cl.Ca).

In canine cardiac myocytes, both delayed-rectifier K⁺ currents and I_to (I_to1 and I_to2) have been demonstrated to play an important role in myocardial action potential repolarization. We have recently described a 4-AP-resistant I_to 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 I_to may be significant during the action potential.

	MATERIALS AND METHODS

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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 (I_Na) 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(-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 I_Cl.Ca (or I_to2), 4-AP (5 mM) was used to block I_to1, and CdCl₂ (300 µM) was used to block the Ca²⁺ current (I_Ca).

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 M 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 (R_s) was electrically compensated to minimize the duration of the capacitive transient. After compensation, R_s was 2.1 ± 0.4 M. 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

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I_to 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. I_Na was suppressed by replacement of external Na⁺ with equimolar choline. I_to1 was blocked by 5 mM 4-AP, I_to2 was inhibited with 5 µM ryanodine, and I_Ca 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 I_to was carried by neither I_to1 (since it was 4-AP insensitive) nor by I_to2 (seen in the presence of ryanodine and absence of I_Ca).

Figure 2 shows the effect of the omission of external K⁺ (K⁺_o) on depolarization-induced I_to. 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 I_to sensitive to Ba²⁺ or K ⁺_o omission. To study voltage dependence of the depolarization-activated I_to, myocytes were depolarized to more positive potentials using the same voltage-clamp protocol as in Fig. 1B. Because the activation of the I_to 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 I_to 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 I_to 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).

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 (), Ipeak (), Iss (), and Ih (), 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.

K⁺ _o dependence and Ba²⁺ concentration-dependent inhibition of the depolarization-induced current. The K⁺ selectivity of Ba²⁺-sensitive I_to was further assessed by evaluating the response of the V_Rev to various K⁺_o concentrations ([K⁺]_o). The V_Rev of the I_to was determined by extrapolating the time-dependent peak current to I_h. A 10-fold change in K⁺_o led to a 52 ± 2 mV/decade (n = 6) shift in the V_Rev value (Fig. 5A), in agreement with the prediction of the Nernst equation (60 mV/decade). To determine whether I_peak and I_ss have similar sensitivity to Ba²⁺, the 50% inhibitory concentration (IC₅₀) of Ba²⁺ on I_peak and I_ss 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 I_peak and 4.9 ± 0.2 µM for I_ss (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.

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.

Kinetics of time-dependent I_to.ir. The time dependence of I_to.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 (1 and 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.

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.

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; t, change in time of P1 and P2 interval.

Effects of intracellular Mg²⁺ and spermine on I_to.ir. Intracellular Mg²⁺ (Mg²⁺_i) and polyamines, such as spermine, have been reported to be related to inward rectification property of I_K1 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 I_to.ir was changed after cell dialysis for 10 min. The omission of Mg²⁺_i did not induce a significant change in I_to.ir (Fig. 10). Similarly, spermine inclusion did not significantly change the current amplitude or I-V relation of I_to.ir either. At a voltage step to 40 mV from 80 mV, I_to.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 I_to1 and K⁺_osensitive I_to.ir. All of the above studies of Ba²⁺- or K⁺_o-sensitive I_to.ir were performed in the presence of 5 mM 4-AP, excluding the participation of classical 4-AP-sensitive I_to1. It was of interest to compare directly some of the properties of 4-AP-sensitive I_to1 with I_to.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 I_to1 (C), currents after K⁺_o omission (D), and K⁺_o omission-sensitive currents (E) in a representative cell. 4-AP-sensitive I_to1 (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 I_to1 shows a linear (ohmic) I-V relation and an activation threshold potential at about 20 mV, whereas the K⁺_o-sensitive I_to.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.

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 I_to.ir to the action potential. We evaluated the possible contribution of I_to.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.

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

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In the present study, we have demonstrated that 1) depolarization elicits a 4-AP-resistant I_to 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 V_Rev of the Ba²⁺- and K⁺_o-sensitive current responds to changes in [K⁺]_o. This novel transient outward component (I_to.ir) shares some features (sensitivity to Ba²⁺ addition and K⁺_o removal, inward rectification) with I_K1. Some properties of I_to.ir are close to those of outward current carried by the recently cloned human TWIK-1 channel (18).

Comparison with previously reported I_to. Two types of I_to have been reported in mammalian cardiac myocytes (32, 38, 39). One is 4-AP-sensitive (I_to1), and the another is a Ca²⁺-activated Cl current (I_to2). The threshold potential for I_to1 activation is 30 to 20 mV, and the I-V relation curve is linear (Fig. 10F; see Ref. 21). I_to2 is activated at 30 mV, corresponding to the activation of I_Ca and intracellular Ca²⁺ transient, and the I-V relation curve is bell shaped (38, 39). I_to.ir is apparent at 70 to 60 mV, just positive to the current reversal potential (e.g., E_K), and the I-V relation curve is neither linear nor bell shaped. I_to.ir is present under conditions (5 mM 4-AP, 5 µM ryanodine, and 300 µM Cd²⁺) that fully suppress I_to1 and I_to2 and has biophysical properties that are clearly different from the two latter, well-described currents.