AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 279: H130-H138, 2000;
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Vol. 279, Issue 1, H130-H138, July 2000

Existence of a transient outward K+ current in guinea pig cardiac myocytes

Gui-Rong Li1,2, Baofeng Yang1, Haiying Sun1, and Clive M. Baumgarten2

1 Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada H3C 3J7; and 2 Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A novel transient outward K+ current that exhibits inward-going rectification (Ito.ir) was identified in guinea pig atrial and ventricular myocytes. Ito.ir was insensitive to 4-aminopyridine (4-AP) but was blocked by 200 µmol/l Ba2+ or removal of external K+. The zero current potential shifted 51-53 mV/decade change in external K+. Ito.ir density was twofold greater in ventricular than in atrial myocytes, and biexponential inactivation occurs in both types of myocytes. At -20 mV, the fast inactivation time constants were 7.7 ± 1.8 and 6.1 ± 1.2 ms and the slow inactivation time constants were 85.1 ± 14.8 and 77.3 ± 10.4 ms in ventricular and atrial cells, respectively. The midpoints for steady-state inactivation were -36.4 ± 0.3 and -51.6 ± 0.4 mV, and recovery from inactivation was rapid near the resting potential (time constants = 7.9 ± 1.9 and 8.8 ± 2.1 ms, respectively). Ito.ir was detected in Na+-containing and Na+-free solutions and was not blocked by 20 nmol/l saxitoxin. Action potential clamp revealed that Ito.ir contributed an outward current that activated rapidly on depolarization and inactivated by early phase 2 in both tissues. Although it is well known that 4-AP-sensitive transient outward current is absent in guinea pig, this Ba2+-sensitive and 4-AP-insensitive K+ current has been overlooked.

transient outward potassium current; whole cell patch clamp; repolarization; excitability; action potential configuration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DEPOLARIZATION-ACTIVATED OUTWARD currents have been distinguished in the myocardium on the basis of their time and voltage dependence and their pharmacological sensitivity (1). Delayed rectifier (30) and transient outward currents (Ito) (9-11) are observed. Rapid and slow delayed rectifier K+ currents (IKr and IKs, respectively) are found in guinea pig (31, 32), dog (37), and human (22) cardiac myocytes. Ultrarapid delayed rectifier K+ currents (IKur or Iss) also are present in rat (4), dog (38), and human (36) atrial cells.

Ito was identified with whole cell voltage-clamp techniques in cardiac cells from a wide range of species, including the rat (8, 17), rabbit (10), dog (24, 35, 37), elephant seal (27), ferret (5), and human (2, 21). Kenyon and Gibbons (18) reported that 4-aminopyridine (4-AP) decreased Ito in sheep cardiac Purkinje fibers. Subsequently, 4-AP has been 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 (7) and rabbit (40, 41) and dog (35, 37) cardiac myocytes. The 4-AP-sensitive and 4-AP-resistant components often are termed Ito1 and Ito2, respectively, after Tseng and Hoffman (35), and Ito2 is a Ca2+-activated transient outward Cl- current (ICl.Ca) (37, 40, 41). Ito1 and Ito2 play important roles in the repolarization of the cardiac action potential (1, 2, 9, 10, 17, 21, 37).

We recently described inward-going rectifying Ito (Ito.ir) in dog ventricular cells (23). Ito.ir is another 4-AP-insensitive transient outward current that is carried by K+, exhibits inward-going rectification, and is blocked by low concentrations of Ba2+. The biophysical and pharmacological properties of Ito.ir are distinct from those of the classic Ito1 and Ito2 (35, 37, 40, 41). To document whether Ito.ir is present in other species and in atria, we studied guinea pig cardiac myocytes. Classic Ito is not present in the guinea pig under physiological conditions (8, 12, 13, 15), and IKr and IKs are thought to be responsible for repolarization of the action potential in the absence of Ito (39). We found that Ito.ir also is present in guinea pig atrial and ventricular cells. Ito.ir is likely to contribute to repolarization and may be more broadly distributed than previously recognized.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation. Single atrial and ventricular myocytes were isolated using a modification of a procedure described previously (20). Briefly, guinea pigs were killed by cervical dislocation, and the heart was quickly removed and placed in oxygenated Tyrode solution. The heart was mounted on a Langendorff column and initially perfused with oxygenated Tyrode solution (37°C, ~5 min) to wash out the blood. After perfusion with Ca2+-free Tyrode solution for 5-8 min, the heart was enzymatically digested with a solution containing 0.06% collagenase (type II, Worthington Biochemical) and 0.1% BSA (Sigma Chemical) until it softened and shed cells. The left atrium and/or ventricle were excised and placed in a high-K+ storage medium (see Solutions). Small aliquots of the solution containing isolated cells were placed in an open perfusion chamber (1 ml) mounted on the stage of an inverted microscope. Experiments were conducted at room temperature (22°C), except as noted for action potential recordings. Only quiescent rod-shaped cells showing clear cross striations were used.

Solutions. Action potentials were recorded from myocytes bathed in Tyrode solution, which contained (in mmol/l) 136 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES; pH was adjusted to 7.4 with NaOH. Voltage-clamp studies were conducted in K+-containing and K+-free bathing solutions designed to isolate currents of interest. NaCl in Tyrode solution was replaced by equimolar choline chloride to block the Na+ current, except where indicated (see Figs. 8 and 9). Atropine (1 µmol/l) was added to block possible muscarinic receptor-mediated current activated by choline. In addition, 200 µmol/l Cd2+ was used to block Ca2+ currents, 5 µmol/l E-4031 to block IKr, and 10 µmol/l 293B to block IKs (3). The K+-free version was made by also omitting KCl. The pipette solution contained (in mmol/l) 20 KCl, 110 potassium aspartate, 1 MgCl2, 10 HEPES, 5 EGTA, 0.1 GTP, and 5 Mg2ATP; pH was adjusted to 7.2 with KOH. The high-K+ storage medium contained (in mmol/l) 20 KCl, 10 KH2PO4, 70 potassium glutamate, 20 taurine, 10 beta -hydroxybutyric acid, 25 glucose, 20 mannitol, and 5 EGTA and 0.1% albumin; pH was adjusted to 7.2 with KOH.

Electrophysiology and data analysis. Membrane currents and action potentials were recorded with the tight-seal whole cell patch-clamp technique with use of an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Data were acquired and command pulses were generated by analog-to-digital and digital-to-analog converters (12-bit) controlled by pClamp 6 software (Axon). Recordings were low-pass filtered at 2 kHz and stored on the hard disk of an IBM-compatible computer.

Borosilicate glass (1.0 mm OD) pipettes were prepared (model P87, Sutter Instruments) to give a resistance of 2-3 MOmega when the pipettes were filled with pipette solution. After the pipette was zeroed in bath solution, a gigaseal was obtained, and the cell membrane was ruptured by gentle suction to establish the whole cell configuration. The liquid junctional potentials were determined by immersing the pipette into the bath filled with pipette solution, zeroing the voltage reading, and switching to the bath solution used for voltage clamp. The average liquid junction potential was 10.6 ± 0.3 mV (n = 14 pipettes) and was not corrected in the experiments, except as noted.

The membrane capacitance was determined by integrating the capacitive response to a 5-mV hyperpolarizing pulse from a holding potential of -60 mV and dividing by the voltage drop. Membrane capacitance was 105 ± 8 and 44 ± 3 pF in ventricular (n = 25) and atrial (n = 21) cells, respectively. Series resistance was electronically compensated.

Nonlinear curve-fitting techniques (Clampfit in pClamp and Sigmaplot, SPSS, Chicago, IL) 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. ANOVA was used for multiple groups. P < 0.05 was considered to indicate significance. Group data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transient outward current in guinea pig ventricular and atrial myocytes. To study whether Ito is present in guinea pig ventricular cells, membrane currents were elicited by 200-ms depolarizing voltage steps from a holding potential of -80 mV to test potentials between -70 and 0 mV in 10-mV increments; steps were applied every 10 s. Figure 1A shows that depolarization elicited a transient outward current at -60 mV and more positive potentials. This current was sensitive to external K+ (Ko+) and was fully abolished by the removal of Ko+ (Fig. 1B). Only membrane capacitive transients and small leak currents were detected in K+-free bathing media. The family of Ko+-sensitive currents, which were obtained by subtracting currents before and after removal of Ko+, are shown in Fig. 1C and at higher gain in Fig. 1D. The original (Fig. 1A) and the Ko+-sensitive currents undergo voltage-dependent inactivation. Moreover, the current inactivation appears to be more rapid at more positive potentials, producing a characteristic crossover of the current traces. Finally, the magnitude of the transient current appears to saturate at positive voltages, indicating inward-going rectification (see Fig. 4B). Similar results were obtained in 21 of 27 ventricular myocytes. The characteristics of the transient outward current in guinea pig ventricular myocytes are similar to those of Ito.ir described previously in dog ventricular cells (23). All the K+-sensitive current is not Ito.ir, however. The inward rectifier, IK1, should not be affected by the cocktail of ion channel blockers employed, and therefore the steady-state K+-sensitive current is expected, at least in part, to represent IK1.


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  		Fig. 1.   Transient outward currents in a ventricular myocyte in Na+-free bathing media containing 5 µmol/l E-4031 to block rapid delayed rectifier K+ current (IKr), 10 µmol/l 293B to block slow delayed rectifier K+ current (IKs), 200 µmol/l Cd2+ to block Ca2+ current (ICa), and 1 µmol/l atropine to block any muscarinic receptor-mediated current activated by choline. A: currents elicited by 200-ms depolarizations from -80 mV to test potentials between -70 and 0 mV. Some of the negative-going capacity spike was omitted to optimize presentation of the currents (voltage-clamp protocol shown in inset of B; calibration in B applies to A - C). B: omission of extracellular K+ (Ko+) abolished the transient outward current and decreased the holding current. Only capacitive transients and a small leak current remained. C: Ko+-sensitive currents obtained by subtracting currents before and after removal of Ko+. D: crossover of inactivating current traces and inward rectification of transient current (same currents as in C) at higher gain and time resolution. Arrows, zero current level.

Ito.ir in dog ventricle is inhibited by low concentrations of Ba2+ (23). Consistent with this result, the transient outward current in guinea pig ventricular myocytes also was sensitive to Ba2+ and was fully blocked by 200 µmol/l Ba2+ (n = 10 cells; data not shown).

To examine whether Ito.ir also is expressed in guinea pig atrial cells, we used the same protocol used in ventricular cells. Figure 2A displays a family of transient outward currents elicited in an atrial myocyte. As in the ventricle, the current was sensitive to K+ and abolished by omission of Ko+ (Fig. 2B). The Ko+-sensitive currents are displayed in Fig. 2C and at higher gain in Fig. 2D. As was the case for Ko+-sensitive transient outward current in ventricular cells, the transient outward current in atrial cells also undergoes voltage-dependent inactivation and exhibits inward rectification. Similar results were obtained in 17 of 25 atrial cells, indicating that Ito.ir is present throughout the working myocardium of the guinea pig.


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  		Fig. 2.   Transient outward currents in an atrial myocyte under conditions described in Fig. 1 legend. A: currents elicited by 200-ms depolarizations from -80 mV to test potentials between -70 and 0 mV. Some of the negative-going capacity spike was omitted to optimize presentation of the currents (voltage-clamp protocol shown in inset of B; calibration in B applies to A - C). B: omission of Ko+ abolished the transient outward current and decreased the holding current. Only capacitive transients and a small leak current remained. C: Ko+-sensitive currents obtained by subtracting currents before and after removal of Ko+. D: crossover of inactivating current traces and inward rectification of transient current (same currents as in C) at higher gain and time resolution. Arrows, zero current level.

Effect of 4-AP on transient outward current. It is well known that Ito1 is sensitive to 4-AP. To test whether the K+-sensitive transient outward current in the guinea pig is sensitive to 4-AP, membrane currents were measured in the absence and presence of 5 mmol/l 4-AP. Figure 3 displays the Ko+-sensitive difference current before (A) and after (B) the application of 4-AP in a ventricular myocyte. A comparison of the traces indicates that the transient outward current was essentially unaffected by 4-AP. This is more clearly shown in Fig. 3C, in which the 4-AP-sensitive difference currents are plotted. For a 200-ms voltage step from -80 to -20 mV, for example, Ito.ir measured as the peak minus the quasi-steady-state K+-sensitive current was 4.1 ± 0.5 pA/pF in control and 4.2 ± 0.6 pA/pF after the application of 5 mmol/l 4-AP (n = 5, P = NS). Similarly, 4-AP did not affect the transient outward current in the four atrial cells tested. 4-AP resistance is a characteristic of the K+ current Ito.ir in canine myocytes (23) but is distinct from the pharmacology of the classic Ito1.


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  		Fig. 3.   Effect of 4-aminopyridine (4-AP) on Ko+-sensitive transient outward K+ current that exhibits inward-going rectification (Ito.ir) in a ventricular cell. A: K+-sensitive current under control conditions recorded as described in Fig. 1C legend. B: K+-sensitive current after application of 5 mmol/l 4-AP for 10 min. Inset: voltage-clamp protocol. C: 4-AP-sensitive current obtained as difference between difference currents in A and B. Small residual is largely due to minor changes in membrane current and electrode properties over time and was not consistent from cell to cell. Arrows, zero current level.

Current-voltage relationship of Ito.ir. The current-voltage (I-V) relationship of Ito.ir was assessed in atrial and ventricular myocytes, as illustrated in Fig. 4A with a recording from a ventricular cell. Ito.ir was taken as the transient component of K+-sensitive membrane current (peak minus quasi-steady-state current at 200 ms), and ISS, the steady-state current, was measured from the zero current level, as was the holding current (Ihold). Currents were expressed as current densities normalized by membrane capacitance to control for variation in cell size and allow more direct comparison results from atrial and ventricular cells.


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  		Fig. 4.   Current-voltage (I-V) relationship of Ko+-sensitive currents. A: schematic showing analysis of Ko+-sensitive current from a ventricular cell recorded as described in Fig. 1C legend with a 200-ms step to a test potential (TP) of -20 mV from a holding potential of -80 mV. Iss, steady-state current; Ihold, holding current. B: I-V relationships for time-dependent component defined as Ito.ir and ISS in ventricular (n = 19) and atrial cells (n = 17). Ito.ir and ISS densities were significantly smaller in atrial than in ventricular cells (P < 0.01). Ko+-sensitive current was obtained as described in legends of Figs. 1C and 2C. Values are means ± SE.

Figure 4B shows the I-V relationships for the Ko+-sensitive currents in ventricular (n = 19) and atrial (n = 17) cells. The density of Ito.ir increased on depolarization, reaching a plateau at potentials positive to -20 mV. The I-V curve for ISS displayed even stronger inward rectification; ISS decreased at potentials positive to -30 mV. This behavior is similar to that of IK1 and suggests that ISS is largely IK1. The possibility that the channels responsible for Ito.ir do not fully inactivate and contribute to ISS is considered below. Although the shapes of the Ito.ir and ISS I-V curves were similar in atrial and ventricular cells, the current densities were significantly greater in ventricular cells.

Ito.ir is most likely a K+ current on the basis of the fact that the current was outward at all potentials positive to K+ equilibrium potential (EK) and that it disappeared on removal of Ko+. When the I-V curves were extrapolated to the voltage axis, the zero current potential for Ito.ir was -69 mV in ventricular cells and -64 mV in atrial cells. On correction for the 10.6-mV liquid junction potential, the zero current potentials were -79.6 mV in ventricular cells and -74.6 mV in atrial cells, close to the calculated EK of -81.2 mV. Moreover, a 10-fold change in Ko+ led to a 53 ± 3 mV/decade (n = 6) shift in the zero current potential in ventricular cells and a 51 ± 2 mV/decade (n = 5) shift in atrial cells, in agreement with the prediction of the Nernst equation. Taken together, these data suggest that Ito.ir is carried by K+ in guinea pig myocytes as in dog ventricular cells (23).

Kinetics of Ito.ir. The kinetics of Ito.ir inactivation were determined by analyzing the Ko+-sensitive current. Figure 5A shows the difference current obtained for a 200-ms voltage step from -80 to -20 mV in a ventricular cell. The transient component was best fit by a biexponential function with time constants of 7.8 and 85.2 ms. The voltage dependence of the time constants in atrial (n = 12) and ventricular (n = 16) cells is shown in Fig. 5B. The fast inactivation time constant (tau 1) was voltage dependent (P < 0.01) in both cell types, and rate of rapid inactivation increased on depolarization. The voltage dependence of tau 1, combined with the depolarization-induced increase in the amplitude of Ito.ir (Fig. 4B), explains the crossover of the current traces when a family of currents is recorded (Figs. 1 and 2). In contrast to tau 1, the slow inactivation time constant (tau 2) was not significantly affected by voltage. Both tau 1 and tau 2 were slightly faster in atrial than in ventricular cells, but the difference was not statistically significant. At a test potential of -20 mV, the fast component accounted for 75% of inactivation in atrial and ventricular myocytes. The voltage dependence of the fraction of current, A1, that inactivates with tau 1 is presented in Fig. 5C. A1 significantly increased on depolarization as tau 1 became faster.


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  		Fig. 5.   Inactivation of Ito.ir is voltage dependent. A: inactivation of Ko+-sensitive Ito.ir in ventricular cell during a 200-ms depolarization from -80 mV to a test potential of -20 mV was fitted by a biexponential function with time constants of 7.8 and 85.2 ms. Inset: voltage-clamp protocol. B: rapid and slow time constants (tau 1 and tau 2) of Ito.ir inactivation obtained as illustrated in A for atrial (n = 12) and ventricular (n = 16) cells. Both tau 1 and tau 2 were slightly smaller in atrial than in ventricular cells. Voltage dependence of tau 1 was significant (P < 0.01), and this component of inactivation became faster at more positive potentials in atrial and ventricular cells. In contrast, tau 2 was not significantly affected by voltage. C: fractional amplitude of current decaying in the fast component (A1/Atotal) plotted as a function of voltage. The fraction of current decaying rapidly significantly increased at more positive potentials (P < 0.01) in both types of myocytes. Ko+-sensitive current was obtained as described in legends of Figs. 1C and 2C. Values are means ± SE.

The voltage dependence of steady-state inactivation of the conductance underlying Ko+-sensitive Ito.ir was determined as illustrated in Fig. 6A. Prepulses, 300 ms or, in some cases, 1,000 ms in duration, were applied at conditioning potentials between -110 and 0 mV, and then currents were recorded during 200-ms test pulses to +10 mV before and after removal of Ko+. The conductance inactivation variable was calculated by dividing Ito.ir at a given prepulse potential by the maximum Ito.ir, and the results with 300- and 1,000-ms prepulses were indistinguishable. Figure 6B shows the voltage dependence of inactivation of conductance and fits to Boltzmann distributions. The half-inactivation voltage (V0.5) was more negative in atrial cells, -51.6 ± 0.4 mV (n = 10 cells), than in ventricular cells, -36.4 ± 0.3 mV (n = 12 cells, P < 0.05), whereas the slope factors, 13.4 ± 0.3 and 12.1 ± 0.3 mV, respectively, were not distinguishable. The different V0.5 values may reflect intrinsic differences between the underlying channels in atrial and ventricular myocytes.


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  		Fig. 6.   Voltage dependence of steady-state inactivation of Ito.ir. A: protocol and representative Ko+-sensitive currents used to assess the voltage dependence of steady-state inactivation of Ito.ir in a ventricular cell. Inset: voltage-clamp protocol. B: currents (I) recorded at +10 mV after 300-ms steps to the condition potential (CP) were normalized by the maximum current obtained (Imax). Data were fit to the Boltzmann relationship: I/Imax = {1 + exp[(V - V0.5)/S]}-1, where V is the conditioning potential, V0.5 is the potential for half-maximal inactivation, and S is the slope factor. The midpoint of the inactivation curve was more negative in atrial (n = 10) than in ventricular cells (n = 12, P < 0.05). Ko+-sensitive current was obtained as described in legends of Figs. 1C and 2C. Values are means ± SE.

The time dependence of recovery from inactivation of Ko+-sensitive Ito.ir was studied with the paired-pulse protocol illustrated in Fig. 7A. Identical 200-ms pulses (P1 and P2) from -80 to -20 mV were delivered every 10 s with varying intervals between P1 and P2. The current during P2 (I2) relative to the current during P1 (I1) is plotted as a function of the P1-P2 recovery interval in Fig. 7B. The recovery of Ito.ir was well fit by monoexponential functions with time constants of 8.8 ± 2.1 and 7.9 ± 1.9 ms in atrial (n = 11) and ventricular (n = 10) cells, respectively (P = NS). Thus recovery from inactivation was rapid at diastolic potentials.


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  		Fig. 7.   Time dependence of recovery from inactivation of Ito.ir. A: representative K+-sensitive currents in ventricular cell and pair-pulse protocol used to assess recovery from inactivation. Two identical 200-ms pulses from -80 to -20 mV (P1 and P2) were applied with a varying P1-P2 interval (Delta t). Inset: voltage-clamp protocol. B: time dependence of recovery (I2/I1) was best fit by monoexponential functions with time constants of 8.7 ms for atrial cells (n = 11) and 8.1 ms for ventricular (n = 10) cells.

Effect of intracellular Mg2+ and spermine on Ito.ir. Intracellular Mg2+ (Mgi2+) and polyamines, such as spermine, are responsible for inward rectification of native IK1 and cloned channels that exhibit inward rectification (16, 26, 29). A decrease in Mgi2+ reduces the extent of inward rectification and thereby increases the amplitude of outward current at positive potentials, whereas addition of polyamines has the opposite effect (29). To study whether Mgi2+ or spermine modulates Ito.ir, we omitted Mgi2+ or included 10 µM spermine in the pipette solution and observed whether Ito.ir changed during cell dialysis. For a voltage step from -80 to -20 mV, Ito.ir recorded immediately after membrane rupture was 4.5 ± 0.6 and 2.1 ± 0.3 pA/pF in ventricular (n = 4) and atrial (n = 4) myocytes, respectively, and 4.6 ± 0.5 and 2.3 ± 0.4 pA/pF after 10 min of dialysis of Mg2+-free pipette solution. In five ventricular and four atrial cells, the inclusion of 10 µM spermine in pipette solution also did not affect Ito.ir. These results were similar to those previously obtained in dog ventricular myocytes (23).

Ito.ir in the presence of external Na+. As described above, Ito.ir was determined in the absence of external Na+, a nonphysiological condition. Because Inoue and Imanaga (15) also described a transient outward K+ current in guinea pig ventricular myocytes under a nonphysiological condition, removing external Ca2+, it is important to clarify whether Ito.ir can be observed in the presence of external Na+.

Recordings of Ito.ir in a ventricular myocyte in the presence of Na+ are shown in Fig. 8. Figure 8A shows a transient Ito.ir at -60 and -50 mV, but Ito.ir was obscured by the Na+ spike at -40 and -30 mV. Figure 8B displays Ito.ir after Na+ channels were blocked with 20 nmol/l saxitoxin (STX); Ito.ir was observed over the entire voltage range examined. Similar results were obtained in a total of four cells. Ito.ir at -50 mV was 2.3 ± 0.2 pA/pF in control and 2.4 ± 0.3 pA/pF after STX (P = NS). The results indicate that activation of Ito.ir does not require removal of external Na+ and that the current is not K+ efflux through Na+ channels.


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  		Fig. 8.   Ito.ir recorded in the presence of external Na+. A: membrane currents activated by 200-ms voltage steps between -70 and -30 mV from a holding potential of -80 mV. Ito.ir was observed at -60 and -50 mV, and the Na+ spike was seen at -40 and -30 mV. B: Na+ channel blocker saxitoxin (STX, 20 nmol/l) inhibited the Na+ spike but did not affect Ito.ir. Traces in A and B are currents in Na+- and K+-containing solutions with and without STX after subtraction of the current in Na+-containing, K+-free solution with 20 nmol/l STX. Similar results were obtained in a total of 4 ventricular myocytes.

Contribution of Ito.ir to the action potential. To gain insight into Ito.ir during the action potential and its contribution to repolarization, we used the action potential-clamp technique to measure Ito.ir. Representative action potentials were recorded from atrial and ventricular cells in normal Tyrode solution at 36°C under current-clamp conditions (stimulation rate 0.5 Hz). These waveforms were latter used as the voltage-clamp command voltage to elicit membrane currents under the conditions previously used to record Ito.ir with square voltage pulses.

Figure 9 illustrates an experiment on a ventricular cell. Figure 9A shows the action potential waveform, and Fig. 9B displays the currents recorded in response to the action potential clamp before and after the removal of Ko+. The Ko+-sensitive current, obtained by subtraction, is shown in Fig. 9C. Two transient components of Ko+-sensitive current were revealed. One transient is evident immediately after depolarization, is thought to result from Ito.ir, and is expected to contribute to early repolarization. A second transient occurs during phase 3 repolarization and corresponds to the contribution of IK1. A small current also was present during the plateau of the action potential. In view of the positive plateau voltage and voltage dependence of steady-state inactivation (Fig. 6), the Ko+-sensitive current during the plateau cannot be attributed to Ito.ir. Similar results were obtained in five of seven ventricular myocytes and, in response to an atrial action potential waveform, in five of nine atrial myocytes (data not shown).


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  		Fig. 9.   Contribution of Ito.ir to the action potential. A: action potential recorded from a guinea pig ventricular myocyte was used to clamp the cell. B: currents elicited by the action potential waveform before (control) and after removal of Ko+ (K+-free). C: Ko+-sensitive current during the action potential, obtained by subtracting membrane currents before and after removal of Ko+. The early transient outward current is consistent with the properties of Ito.ir, as determined from square voltage steps; the transient current during phase 3 is likely to primarily reflect the inward rectifier IK1. Ko+-sensitive current was obtained as described in legends of Figs. 1C and 2C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrated that Ito.ir, a transient outward K+ current that undergoes inward rectification, is present in guinea pig atrial and ventricular myocytes. Ito.ir inactivated and recovered from inactivation very quickly, was fully suppressed by the removal of Ko+ or by Ba2+ (200 µmol/l), and was insensitive to 4-AP (5 mmol/l). The current is most likely a K+ current, because it was outward over a wide range of voltage positive to EK, and the zero current potential shifted 51-53 mV/decade in response to altered Ko+. Ito.ir is not K+ efflux via Na+ channels, because it was not blocked by STX. Action potential-clamp experiments showed that Ito.ir generated a significant outward current at the time of phase 1 repolarization. Although the current density was less in atrial than in ventricular myocytes, Ito.ir is expected to affect the action potential configuration of both types of cells. Ito.ir also is present in canine (23) as well as in human and rabbit (unpublished observations) ventricular myocytes. This indicates that Ito.ir is widespread in mammalian hearts as well as in atria and ventricles.

Ito.ir is a novel K+ current. The biophysical and pharmacological properties of Ito.ir were distinct from those of the two types of Ito that have been well studied in mammalian cardiac myocytes (35, 37, 40, 41). Ito1 is a 4-AP-sensitive K+ current, and Ito2 is Ca2+-activated Cl- current. The threshold potential for Ito1 activation is -20 or -30 mV, and its I-V relationship is linear (2, 21, 23, 37). Ito2 activates near -30 mV, corresponding to the activation of ICa and the intracellular Ca2+ transient, and its I-V relationship is bell shaped (37, 40, 41). In contrast, Ito.ir was apparent at -60 mV in guinea pig cardiac myocytes, and its I-V relationship was neither linear nor bell shaped. Moreover, Ito.ir was not sensitive to the Ito1 blocker 4-AP and was present under conditions (5 mmol/l EGTA in pipette solution and 200 µmol/l Cd2+ in superfusate) that fully suppress Ito2. In view of these biophysical and pharmacological differences, it is reasonable to suggest that an ion channel distinct from those underlying Ito1 and Ito2 is responsible for Ito.ir.

Martin et al. (25) reported a 3,4-diaminopyridine-insensitive, Ca2+-independent transient outward K+ current in feline cardiac ventricular myocytes. This current was not initially observed on establishment of whole cell conditions; its activation depended on the duration of dialysis, and therefore, it was named the patch-duration-dependent K+ current, IK(PDD). IK(PDD) shares some properties with Ito.ir, including 4-AP insensitivity, Ba2+ sensitivity, and voltage-dependent inactivation. In contrast to IK(PDD), Ito.ir was present immediately on membrane rupture, and its inwardly rectifying I-V relationship was clearly different from the I-V relationship for IK(PDD), which is linear between -40 and +60 mV.

A transient outward K+ current previously was described in the guinea pig only after removal of external Ca2+, a nonphysiological condition (15). This and our experiments under Na+-free conditions raised the possibility that Ito.ir was only present under conditions not relevant to normal function. However, Fig. 8 shows that Ito.ir can be observed under physiological conditions in the presence of Ca2+ and Na+, and it does not reflect K+ efflux through Na+ channels.

Although IKr exhibits inward rectification at positive potentials (31, 32), it is unlikely that IKr contributed to the present observations. In contrast to Ito.ir, IKr has a "bell-shaped" I-V relationship (31, 32). Furthermore, Ito.ir was studied in the presence of the IKr channel blocker E-4031 (5 µmol/l). E-4031 is an highly effective IKr blocker (31, 32), and the block of IKr is voltage and time independent (6). Therefore, it is unlikely that Ito.ir reflects time- and voltage-dependent "unblock" of IKr.

Is Ito.ir a component of IK1? Several characteristics of Ito.ir in the guinea pig and dog are similar to characteristics of IK1: both currents are blocked by low concentrations of Ba2+ and by removal of Ko+, and both display inward-going rectification. Therefore, it is important to consider whether Ito.ir could be a component of IK1. One might imagine, for example, that the decay of Ito.ir could be due to the voltage-dependent block of IK1 by Mgi2+ that gives rise to rectification (16, 26). This idea seems unlikely. At only 500 µmol/l Mgi2+, a concentration lower than that present here, IK1 rectification on depolarization appears to be instantaneous in guinea pig myocytes (16). Furthermore, omitting Mg2+ or adding spermine to the pipette solution did not modulate Ito.ir, whereas outward IK1 should have been increased with Mg2+-free pipette solution (16, 26) and depressed by spermine (29). A selective blocker that distinguishes between IK1 and Ito.ir would further clarify this question, but such an agent has not been identified.

In the absence of a blocker that distinguishes between IK1 and Ito.ir, we empirically defined Ito.ir as the transient component of Ko+-sensitive current remaining after other outward K+ currents were blocked. However, we cannot rigorously exclude the possibility that a rapidly inactivating component of IK1 contaminated the currents measured as Ito.ir. In theory, this might have affected the shape of the Ito.ir I-V curve (Fig. 4B) and the analyses of its kinetics. The presence of IK1 also made it difficult to measure the reversal potential of Ito.ir with use of deactivating tail currents. We therefore estimated the reversal potential as the zero current potential for Ito.ir by extrapolating the I-V curves to the voltage axis. This approach can be criticized because the voltage dependence of activation might influence the zero current potential. Nevertheless, other factors strongly support the conclusion that Ito.ir is largely a K+ current. Not only was Ito.ir absent in K+-free bathing media, but also the zero current potential shifts 51-53 mV/decade change in Ko+. Furthermore, Ito.ir is outward over a wide range of potentials from near EK to +40 mV. No other ion can readily explain the direction of current flow.

Physiological implications. In view of its rapid activation and inactivation, Ito.ir should make an important contribution to K+ efflux during phase 1, especially in guinea pig myocytes in which 4-AP-sensitive Ito is absent (8, 12). Previously, repolarization in the guinea pig largely has been attributed to the delayed rectifier K+ currents (13, 39), a conclusion that now should be revisited. The rapid recovery of Ito.ir from inactivation suggests that its contribution to the action potential should be independent of heart rate. The activation of Ito.ir (Fig. 4B) and steady-state inactivation curve (Fig. 6B) overlapped between -60 and 0 mV. This implies that a fraction of Ito.ir, up to 10 to 20% of peak current, does not inactivate over this voltage range. Because the plateau of the guinea pig action potential is positive to 0 mV, the potential at which rapid and complete inactivation occurs, noninactivating Ito.ir should have little direct effect during the plateau or on action potential duration. On the other hand, a noninactivating, Ba2+-sensitive K+ current arising from Ito.ir might easily be mistaken for IK1 in voltage-clamp experiments, and we suspect that the noninactivating component of Ito.ir contributed to the steady-state K+-sensitive current (Fig. 4B, ISS).

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 (14, 33) by stabilizing the resting potential (29). Because significant outward currents carried by Ito.ir can be elicited by depolarization at very negative potentials over a time course comparable to INa, Ito.ir also may play a role in excitability. The density of Ito.ir was lower and the voltage-dependent inactivation (V0.5) of Ito.ir was more negative in atrial than in ventricular cells. Whether these differences contribute to electrophysiological variation between atrial and ventricular cells remains to be studied.

Molecular identity. The molecular identity of Ito.ir is unknown. In contrast, much has been learned about the proteins responsible for Ito1. The preponderance of the evidence links Ito1 with Kv4.3 in humans and perhaps the rat, with Kv4.2 in the rat and mouse, and with Kv1.4 in the rabbit and perhaps the ferret. In addition, Kv4.x and Kv1.4 may not be uniformly distributed across the ventricular wall, giving rise to fast and slow components of Ito1 (for review see Refs. 28 and 34). We are unaware of studies demonstrating the expression of these genes in guinea pig myocytes. Several other cloned K+ channels also generate transient outward currents on expression. Some properties of Ito.ir are similar to those of the outward current carried by the recently cloned human TWIK-1 K+ channel, which is expressed predominantly in the heart and brain (19). Currents corresponding to the behavior of TWIK-1 have not been identified in native cells. Expression of TWIK-1 in oocytes gives rise to a fast activating and rapidly decaying transient outward current that displays inward rectification over a voltage range similar to Ito.ir. TWIK-1 current is blocked by Ba2+ (IC50 = 100 µmol/l) and insensitive to 4-AP (19), properties also shared by Ito.ir. On the other hand, TWIK-1 produces a noninactivating current over a wide range of negative potentials, whereas Ito.ir activated positive to -70 mV. This apparent difference may not be real. Because Ito.ir was defined as the transient component of current, any noninactivating component was included in ISS.

In conclusion, a novel transient outward current, Ito.ir, is present in guinea pig atrial and ventricular myocytes as well as in dog myocytes. Ito.ir is likely to contribute to phase 1 repolarization. Because it is insensitive to 4-AP and sensitive to low concentrations of Ba2+, Ito.ir may easily be confused with other membrane currents. Further work is required to fully understand the physiological role of Ito.ir in various species and to identify the molecular entity responsible for this current.


    ACKNOWLEDGEMENTS

This work was supported by the Heart and Stroke Foundation of Québec, Fonds de la Recherche en Santé du Québec, the Fonds de Recherche de l'Institut de Cardiologie de Montréal, and National Heart, Lung, and Blood Institute Grant HL-46764. G.-R. Li was a research scholar of the Fonds de la Recherche en Santé du Québec when most of the experiments were performed.


    FOOTNOTES

Address for reprint requests and other correspondence: G.-R. Li, Dept. of Physiology, Box 980551, Medical College of Virginia, 1101 E. Marshall St., Richmond, VA 23298-0551 (E-mail: gli{at}hsc.vcu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 18 August 1999; accepted in final form 7 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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