Journal of Physiology (1991), 435, pp. 395-420 With 17 figures Printed in Great Britain
395
THE TRANSIENT K+ CURRENT IN RAT VENTRICULAR MYOCYTES: EVALUATION OF ITS Ca2+ AND Na+ DEPENDENCE
BY IAIN D. DUKES* AND MARTIN MORADt From the Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104-6085, USA
(Received 24 April 1990) SUMMARY
1. The transient outward K+ current (Ito) was studied in enzymatically isolated rat ventricular myocytes using the whole-cell patch clamp technique. 2. At holding potentials between -100 and -60 mV, depolarizing pulses activated outward current which was composed of transient and maintained components. These components differed from each other in their activation voltage range as well as in their kinetics of inactivation. 3. The transient component, in turn, appeared to be composed of rapidly and slowly inactivating components. Subtraction of ICa from the total current, or nifedipine pre-treatment, eliminated the slowly inactivating component of Ito indicating that the time course of inactivation of Ito may be contaminated by ICa* 4. Reduction of the holding potential from -100 mV to less negative holding potentials reduced all components of Ito, such that at holding potentials of -40 mV, very little or no Ito could be measured. 5. Elevation of [Ca2"]. activated Ito at holding potentials of -40 mV, and substitution of external Ca2+ by Sr2+ suppressed Ito, consistent with findings from other preparations and in support of a Ca2+-activated component of Itk. 6. Elevations of [Ca2+]0, however, also shifted the steady-state activation and inactivation parameters of the transient K+ current, such that a greater proportion of Ito channels were activated at the less negative holding potentials. 7. The shifts in the activation and inactivation parameters of the transient outward current were not mimicked by equivalent changes in external Mg2+. 8. Modulators of Ca2+ release from the sarcoplasmic reticulum (SR) such as caffeine and ryanodine suppressed Ito regardless of whether the myocytes were dialysed with low or high concentrations of Ca2' buffers (EGTA or BAPTA, 0-5-14 mM) or whether nifedipine was used to block ICa9. 4-Aminopyridine (4-AP) blocked Ito in a dose-dependent manner, completely suppressing it at 10 mm. Similarly, tedisamil, a new K+ channel blocker, completely and reversibly blocked It. at 5-20 /,M concentrations. 10. TTX (10 ,tM) or removal of external Na+ decreased Ito, consistent with the idea *
Present address: Glaxo Inc. Research Institute, Research Triangle Park, NC 27709, USA.
t To whom all correspondence should be addressed. MS 8453
396
I. D. DUTKES AND M. MORAD
that a component of Ito was Na+ activated. Both interventions, however, also shifted the voltage dependence of the activation and inactivation of Ito to more negative potentials, such that at - 100 mV neither intervention had a significant effect on Ito. Alterations in [Na+]i had no effect on Ito. 11. We conclude, therefore, that Ito in rat ventricular myocytes is composed of a single voltage-dependent component which is modulated by changes in extracellular but not intracellular Na+ and Ca2+. This modulation is mediated by cation-induced shifts in the gating parameters of Ito. INTRODUCTION
A distinct set of potassium channels that open and close rapidly following depolarization exists in the membranes of most excitable cells. This transient K+ current, first described by Hagiwara, Yoshida & Yoshii (1981) in molluscan neurones, is generally referred to as 'A in neurones and as Ito in cardiac muscle. Ito has been described in a variety of cardiac tissues, but there are widely differing views as to its modulation. Siegelbaum & Tsien (1980) observed a transient outward current in calf Purkinje fibres that was abolished by reduction of external Ca2, substitution of external Ca2+ with Sr2+ and injection of EGTA, leading them to conclude that its activation depended upon elevation of [Ca2+]i. In support of this contention, Maylie & Morad (1984) demonstrated that the transient outward current recorded from elephant seal fibres was abolished by concentrations of caffeine that suppressed phasic tension. Similarly, Sutko & Kenyon (1983) reported that in calf Purkinje fibres caffeine, ryanodine and Sr2+ substitution all reduced the transient outward current. Kenyon & Gibbons (1979) described a second type of It. the activation of which was independent of changes in [Ca2+]i, and was blocked by 025-1 mM-4aminopyridine (4-AP). The existence of a Ca2+-independent Ito was confirmed in sheep Purkinje fibres (Boyett, 1981); in rat ventricular myocytes (Josephson, Sanchez-Chapula & Brown, 1984); in rabbit ventricular muscle (Kukushkin, Gainullin & Sozunov, 1983; Hiraoka & Kawano, 1989); in dog Purkinje fibres (Nakayama, Palfrey & Fozzard, 1989); and in rabbit crista terminalis (Giles & van Ginneken, 1985). Coraboeuf & Carmeliet (1982) suggested that both Ca2+-dependent and independent components were present in sheep Purkinje fibres, and could be dissected on the basis of their differential sensitivity to caffeine and 4-AP. Such co-existence of Ca2+-dependent and independent transient outward currents within the same tissue have been supported by Escande, Coulombe, Faivre, Deroubaix & Coraboeuf (1987) in human atrium, by Giles & Imaizumi (1988) in rabbit atrium and ventricle, by Hiraoka & Kawano (1989) in rabbit ventricle and most recently by Tseng & Hoffman (1989) in canine ventricle. To further complicate matters, a component of It. and 'A in both neurones (Bader, Bernheim & Bertrand, 1985; Dryer, Fujii & Martin, 1989) and cardiac muscle (Dukes & Morad, 1987) appears to be attenuated by tetrodotoxin (TTX) or removal of external Na+. On the basis of these findings an additional Na+-activated component of transient outward current was postulated (Bader et al. 1985; Dukes & Morad, 1987).
TRANSIENT OUTH"ARD CURRE.NT IN RAT MYOCYTES
397 In this we report we have re-examined the evidence for the voltage-, Ca2+- or Na+dependent gating of the transiently activating K+ current in rat ventricular myocytes. Our experiments support the existence of only one voltage-dependent component of Ito which is strongly modulated by the extracellular concentration of Na+ and Ca2+. METHODS
Isolation of cardiac myocytes Myocardial cells were prepared essentially by using an earlier described method (Mitra & Morad, 1985). Briefly, rats of either sex (weight 200-300 g) were anaesthetized with pentobarbitone, their hearts rapidly removed, washed in zero-Ca2+ Tyrode solution (for composition see Table 1) and then perfused for 5 min at 12 ml min-' with the zero-Ca2" solution in a Langendorff set-up. The heart was then perfused for 20 min with Tyrode solution containing protease type XIV 1 U/ml (Sigma) and collagenase type III 42 U/ml (Sigma). After brief perfusion with a 0-2 mM-Ca2+containing Tyrode solution, the cells were mechanically dispersed. The [Ca2"]. was then increased to 1 8 mm and the suspension of cells transferred to small dishes where they were stored at 5 °C until used. The procedure yielded approximately 80% Ca2+-tolerant cells, which were viable for about 10-12 h.
Experimental approach A small aliquot of cells were transferred to a perfusion chamber (volume 1 ml) which was placed on the stage of an inverted microscope (Diaphot, Nikon) through which the cells were directly observed. Cells were also monitored on a video screen linked to the microscope via an NEC camera. Bathing solutions were introduced at one end of the chamber and removed by suction at the opposite end. Fire-polished electrodes (2-4 MQ) were used to achieve seal resistances in the gigaohm range. The intracellular pipette solutions were primarily KCl based, the composition of which is shown in Table 1. In some experiments, myocytes were dialysed with lower concentrations of EGTA or BAPTA allowing them to contract in order to unmask possible Ca2+-dependent K+ currents. In such mvocvtes. cell shortening was monitored using a photodiode array (NTabauer & Morad, 1990). A variety of external solutions were used, the composition of which are shown in Table 1. Membrane currents were recorded (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) using a Dagan 8900 amplifier and where possible were leak and capacitance subtracted in conjunction with a PCLAMP data acquisition and analysis program (Axon Instruments). Where leak and capacitance subtractions were carried out, one of two experimental protocols was used. In the first, K+ ions were left out of the external solution and 100 /JM-Ba2" was included to eliminate current passing through the inward rectifier. Hyperpolarizing test pulses of 20 mV amplitude, elicited from a holding potential of -60 mV were recorded, scaled and subtracted from the corresponding depolarizing experimental pulses. Alternatively, 20 mV hyperpolarizing pulses were delivered from a holding potential of -20 mV (a voltage at which the inwardly rectifying K+ channels are inactivated in rat myocytes), removing the necessity of excluding K+ from the external solution. The holding potential of the experimental myocytes was regulated between - 100 and -40 mV, depending on the ionic channel investigated. Holding potentials of -100 to -80 mV were used primarily to examine Ito and the sodium current (Na)' while holding potentials between -50 and -40 mV were used to measure the calcium current (ICa). All clamp pulses were delivered at a rate of 0-2 Hz. Vhen only lCa was examined the myocytes were dialysed with 120 mM-Cs'. All experiments were carried out at room temperature, 22-25 °C. RESULTS
Voltage dependence of transient outward current When the ventricular myocytes were voltage clamped at a holding potential of -80 mV, depolarizing clamps up to +60 mV progressively activated outward current (Fig. 1A) which appeared to have multiple components. A transient outward
398
I. D. DUKES AND M. MORAD
current (It.) appeared at voltages positive to -50 mV. Between -50 and -10 mV, Ito decayed completely within 60 ms, whereas at potentials positive to -10 mV, the
transient current was superimposed upon a slowly decaying maintained outward current (Imaintained) Figure 1B shows the current-voltage relation for Ito expressed in three alternate ways. The total outward current was defined as the net outward current after
TABLE 1. Composition of external and internal solutions External solutions (in mM) 135 NaCl, 5-4 KCl, 2 CaCl2, 10 HEPES, 10 glucose, pH 7 4. Control solution 135 N-methyl-D-glucamine chloride (or Tris-Cl or choline Zero-sodium solution chloride), 5-4 KCl, 2 CaCl2, 10 HEPES, 10 glucose, pH 7-4. Low-calcium solution 137 NaCl, 5-4 KCl, 0 5 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 132 NaCl, 5-4 KCl, 5 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 5 mM-calcium solution 127 NaCl, 5-4 KCl, 10 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 10 mM-calcium solution 117 NaCl, 5-4 KCl, 20 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 20 mM-calcium solution Internal solutions 125 KCl, 10 NaCl, 14 EGTA, 1 CaCl2, 10 HEPES, 5 Mg-ATP, Standard solution pH 7-2, pCa 9-6. 136 KCl, 10 NaCl, 1 EGTA (or BAPTA), 0-5 CaCl2, 10 HEPES, Low-buffer solution 10 Mg-ATP, pH 7-2, pCa 7-1. 135 KCl, 14 EGTA, 1 CaCl2, 10 HEPES, 5 Mg-ATP, pH 7-2, Zero-sodium solution pCa 9-6. 115 KCl, 20 NaCl, 14 EGTA, 1 CaCl2, 10 HEPES, 10 Mg-ATP, High-sodium solution pH 7-2, pCa 9-6.
correction for leakage current (Fig. 1B, 0). The transient outward current was derived by subtracting from the total outward current, the maintained current at the end of a 160 ms test pulse (Fig. 1B, A). The maintained component of outward current was defined as the current remaining at the end of a 160 ms test pulse, following leak subtraction (Fig. 1B, 0). It was apparent from these I-V revelations that the activation ranges of the transient and maintained currents were different. Furthermore, evaluation of the I-V relation of the transient current suggested the possible existence of two components of Ito, one showing a bell-shaped voltage dependence with a maximum around -20 mV, and the other with a monotonic voltage dependence in the range + 10 to + 60 mV (Fig. 1B, A/). Consistent with this idea, the time course of the inactivation process was much faster at voltages between -40 and -10 mV (average time constant, 18 ms (n = 3)) compared to more positive potentials (-10 to + 60 mV) (average time constant, 48 ms (n = 3)) in a manner similar to that seen in Purkinje fibres (Coraboeuf & Carmeliet, 1982). To study separately the characteristics of Imaintained and Ito, we investigated the effects of alterations of the holding potential on the two current components. In the holding potential range between -100 and -80 mV little change in It. occurred (compare 0 versus * in Fig. 2D). At holding potentials of -60 mV (Fig. 2B) and -50 mV, however, the transient outward current at +60 mV was reduced by 30 and 70 % (n = 5), respectively, compared to that recorded from the -80 mV holding potential (Fig. 2A). This was reflected in the I-V relation as a decrease in both the
TRANSIENT OUTWARD CURRENT IN RAT MYOCYTES
399
bell-shaped and linear components of It. (Fig. 2D, A and A). At a holding potential of -40 mV, the transient outward current appeared to be completely suppressed revealing the presence of a prominent calcium current (Fig. 2 C). Examination of the I-V relation at this holding potential showed that the bell-shaped region of Ito
A
~~+60 mv
\
*109~~ B
20
m
60
5+
/ 0
4/
0
/(nA) 3
o 0
A
A
A
o/
2-
0 20 60 40 V (mV) Fig. 1. Features of the peak (It.) and maintained ('maintained) components of outward current. A, outward current recorded from a single rat ventricular myocyte from a holding potential of -80 mV, activated in response to depolarizing steps delivered in 10 mV increments up to + 60 mV. The pulse to -70 mV activates no time-dependent current, the pulses to -60 and -50 mV activate only sodium current, while pulses above -50 mV activate increasing amounts of a transient outward current (Ito). At potentials positive to -10 mV a maintained outward current which shows no decay was activated. No net inward calcium current ICa was evident in these current records. Currents were both leak and capacitance subtracted. B, the current-voltage relation for the outward current expressed in three alternative ways: total outward current, following leak subtraction (0), outward current remaining at the end of a 160 ms depolarizing pulse (Imaintained), following leak subtraction (@) and transient outward current (Ito) obtained by subtracting the total and maintained outward currents (A). -60
-40
-20
occurred over the same voltage range as the negative bell-shaped curve of ICa (compare A and E, Fig. 2D). The maintained outward current showed a similar voltage dependence to the transient current as it was unaffected by shifts of holding potentials from -100 to
I. D. DUKES AND M. MORAD
400
-80 mV, but was significantly suppressed at a holding potential of -40 mV (n = 5, data not shown). Thus changes in the holding potential failed to separate either the different components of It. or the transient from the maintained outward current. The biphasic nature of the I-V relation of Ito may result either from two populations of channels activating over separate voltage ranges or alternatively that 500 pA
A ,
395
THE TRANSIENT K+ CURRENT IN RAT VENTRICULAR MYOCYTES: EVALUATION OF ITS Ca2+ AND Na+ DEPENDENCE
BY IAIN D. DUKES* AND MARTIN MORADt From the Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104-6085, USA
(Received 24 April 1990) SUMMARY
1. The transient outward K+ current (Ito) was studied in enzymatically isolated rat ventricular myocytes using the whole-cell patch clamp technique. 2. At holding potentials between -100 and -60 mV, depolarizing pulses activated outward current which was composed of transient and maintained components. These components differed from each other in their activation voltage range as well as in their kinetics of inactivation. 3. The transient component, in turn, appeared to be composed of rapidly and slowly inactivating components. Subtraction of ICa from the total current, or nifedipine pre-treatment, eliminated the slowly inactivating component of Ito indicating that the time course of inactivation of Ito may be contaminated by ICa* 4. Reduction of the holding potential from -100 mV to less negative holding potentials reduced all components of Ito, such that at holding potentials of -40 mV, very little or no Ito could be measured. 5. Elevation of [Ca2"]. activated Ito at holding potentials of -40 mV, and substitution of external Ca2+ by Sr2+ suppressed Ito, consistent with findings from other preparations and in support of a Ca2+-activated component of Itk. 6. Elevations of [Ca2+]0, however, also shifted the steady-state activation and inactivation parameters of the transient K+ current, such that a greater proportion of Ito channels were activated at the less negative holding potentials. 7. The shifts in the activation and inactivation parameters of the transient outward current were not mimicked by equivalent changes in external Mg2+. 8. Modulators of Ca2+ release from the sarcoplasmic reticulum (SR) such as caffeine and ryanodine suppressed Ito regardless of whether the myocytes were dialysed with low or high concentrations of Ca2' buffers (EGTA or BAPTA, 0-5-14 mM) or whether nifedipine was used to block ICa9. 4-Aminopyridine (4-AP) blocked Ito in a dose-dependent manner, completely suppressing it at 10 mm. Similarly, tedisamil, a new K+ channel blocker, completely and reversibly blocked It. at 5-20 /,M concentrations. 10. TTX (10 ,tM) or removal of external Na+ decreased Ito, consistent with the idea *
Present address: Glaxo Inc. Research Institute, Research Triangle Park, NC 27709, USA.
t To whom all correspondence should be addressed. MS 8453
396
I. D. DUTKES AND M. MORAD
that a component of Ito was Na+ activated. Both interventions, however, also shifted the voltage dependence of the activation and inactivation of Ito to more negative potentials, such that at - 100 mV neither intervention had a significant effect on Ito. Alterations in [Na+]i had no effect on Ito. 11. We conclude, therefore, that Ito in rat ventricular myocytes is composed of a single voltage-dependent component which is modulated by changes in extracellular but not intracellular Na+ and Ca2+. This modulation is mediated by cation-induced shifts in the gating parameters of Ito. INTRODUCTION
A distinct set of potassium channels that open and close rapidly following depolarization exists in the membranes of most excitable cells. This transient K+ current, first described by Hagiwara, Yoshida & Yoshii (1981) in molluscan neurones, is generally referred to as 'A in neurones and as Ito in cardiac muscle. Ito has been described in a variety of cardiac tissues, but there are widely differing views as to its modulation. Siegelbaum & Tsien (1980) observed a transient outward current in calf Purkinje fibres that was abolished by reduction of external Ca2, substitution of external Ca2+ with Sr2+ and injection of EGTA, leading them to conclude that its activation depended upon elevation of [Ca2+]i. In support of this contention, Maylie & Morad (1984) demonstrated that the transient outward current recorded from elephant seal fibres was abolished by concentrations of caffeine that suppressed phasic tension. Similarly, Sutko & Kenyon (1983) reported that in calf Purkinje fibres caffeine, ryanodine and Sr2+ substitution all reduced the transient outward current. Kenyon & Gibbons (1979) described a second type of It. the activation of which was independent of changes in [Ca2+]i, and was blocked by 025-1 mM-4aminopyridine (4-AP). The existence of a Ca2+-independent Ito was confirmed in sheep Purkinje fibres (Boyett, 1981); in rat ventricular myocytes (Josephson, Sanchez-Chapula & Brown, 1984); in rabbit ventricular muscle (Kukushkin, Gainullin & Sozunov, 1983; Hiraoka & Kawano, 1989); in dog Purkinje fibres (Nakayama, Palfrey & Fozzard, 1989); and in rabbit crista terminalis (Giles & van Ginneken, 1985). Coraboeuf & Carmeliet (1982) suggested that both Ca2+-dependent and independent components were present in sheep Purkinje fibres, and could be dissected on the basis of their differential sensitivity to caffeine and 4-AP. Such co-existence of Ca2+-dependent and independent transient outward currents within the same tissue have been supported by Escande, Coulombe, Faivre, Deroubaix & Coraboeuf (1987) in human atrium, by Giles & Imaizumi (1988) in rabbit atrium and ventricle, by Hiraoka & Kawano (1989) in rabbit ventricle and most recently by Tseng & Hoffman (1989) in canine ventricle. To further complicate matters, a component of It. and 'A in both neurones (Bader, Bernheim & Bertrand, 1985; Dryer, Fujii & Martin, 1989) and cardiac muscle (Dukes & Morad, 1987) appears to be attenuated by tetrodotoxin (TTX) or removal of external Na+. On the basis of these findings an additional Na+-activated component of transient outward current was postulated (Bader et al. 1985; Dukes & Morad, 1987).
TRANSIENT OUTH"ARD CURRE.NT IN RAT MYOCYTES
397 In this we report we have re-examined the evidence for the voltage-, Ca2+- or Na+dependent gating of the transiently activating K+ current in rat ventricular myocytes. Our experiments support the existence of only one voltage-dependent component of Ito which is strongly modulated by the extracellular concentration of Na+ and Ca2+. METHODS
Isolation of cardiac myocytes Myocardial cells were prepared essentially by using an earlier described method (Mitra & Morad, 1985). Briefly, rats of either sex (weight 200-300 g) were anaesthetized with pentobarbitone, their hearts rapidly removed, washed in zero-Ca2+ Tyrode solution (for composition see Table 1) and then perfused for 5 min at 12 ml min-' with the zero-Ca2" solution in a Langendorff set-up. The heart was then perfused for 20 min with Tyrode solution containing protease type XIV 1 U/ml (Sigma) and collagenase type III 42 U/ml (Sigma). After brief perfusion with a 0-2 mM-Ca2+containing Tyrode solution, the cells were mechanically dispersed. The [Ca2"]. was then increased to 1 8 mm and the suspension of cells transferred to small dishes where they were stored at 5 °C until used. The procedure yielded approximately 80% Ca2+-tolerant cells, which were viable for about 10-12 h.
Experimental approach A small aliquot of cells were transferred to a perfusion chamber (volume 1 ml) which was placed on the stage of an inverted microscope (Diaphot, Nikon) through which the cells were directly observed. Cells were also monitored on a video screen linked to the microscope via an NEC camera. Bathing solutions were introduced at one end of the chamber and removed by suction at the opposite end. Fire-polished electrodes (2-4 MQ) were used to achieve seal resistances in the gigaohm range. The intracellular pipette solutions were primarily KCl based, the composition of which is shown in Table 1. In some experiments, myocytes were dialysed with lower concentrations of EGTA or BAPTA allowing them to contract in order to unmask possible Ca2+-dependent K+ currents. In such mvocvtes. cell shortening was monitored using a photodiode array (NTabauer & Morad, 1990). A variety of external solutions were used, the composition of which are shown in Table 1. Membrane currents were recorded (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) using a Dagan 8900 amplifier and where possible were leak and capacitance subtracted in conjunction with a PCLAMP data acquisition and analysis program (Axon Instruments). Where leak and capacitance subtractions were carried out, one of two experimental protocols was used. In the first, K+ ions were left out of the external solution and 100 /JM-Ba2" was included to eliminate current passing through the inward rectifier. Hyperpolarizing test pulses of 20 mV amplitude, elicited from a holding potential of -60 mV were recorded, scaled and subtracted from the corresponding depolarizing experimental pulses. Alternatively, 20 mV hyperpolarizing pulses were delivered from a holding potential of -20 mV (a voltage at which the inwardly rectifying K+ channels are inactivated in rat myocytes), removing the necessity of excluding K+ from the external solution. The holding potential of the experimental myocytes was regulated between - 100 and -40 mV, depending on the ionic channel investigated. Holding potentials of -100 to -80 mV were used primarily to examine Ito and the sodium current (Na)' while holding potentials between -50 and -40 mV were used to measure the calcium current (ICa). All clamp pulses were delivered at a rate of 0-2 Hz. Vhen only lCa was examined the myocytes were dialysed with 120 mM-Cs'. All experiments were carried out at room temperature, 22-25 °C. RESULTS
Voltage dependence of transient outward current When the ventricular myocytes were voltage clamped at a holding potential of -80 mV, depolarizing clamps up to +60 mV progressively activated outward current (Fig. 1A) which appeared to have multiple components. A transient outward
398
I. D. DUKES AND M. MORAD
current (It.) appeared at voltages positive to -50 mV. Between -50 and -10 mV, Ito decayed completely within 60 ms, whereas at potentials positive to -10 mV, the
transient current was superimposed upon a slowly decaying maintained outward current (Imaintained) Figure 1B shows the current-voltage relation for Ito expressed in three alternate ways. The total outward current was defined as the net outward current after
TABLE 1. Composition of external and internal solutions External solutions (in mM) 135 NaCl, 5-4 KCl, 2 CaCl2, 10 HEPES, 10 glucose, pH 7 4. Control solution 135 N-methyl-D-glucamine chloride (or Tris-Cl or choline Zero-sodium solution chloride), 5-4 KCl, 2 CaCl2, 10 HEPES, 10 glucose, pH 7-4. Low-calcium solution 137 NaCl, 5-4 KCl, 0 5 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 132 NaCl, 5-4 KCl, 5 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 5 mM-calcium solution 127 NaCl, 5-4 KCl, 10 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 10 mM-calcium solution 117 NaCl, 5-4 KCl, 20 CaCl2, 10 HEPES, 10 glucose, pH 7-4. 20 mM-calcium solution Internal solutions 125 KCl, 10 NaCl, 14 EGTA, 1 CaCl2, 10 HEPES, 5 Mg-ATP, Standard solution pH 7-2, pCa 9-6. 136 KCl, 10 NaCl, 1 EGTA (or BAPTA), 0-5 CaCl2, 10 HEPES, Low-buffer solution 10 Mg-ATP, pH 7-2, pCa 7-1. 135 KCl, 14 EGTA, 1 CaCl2, 10 HEPES, 5 Mg-ATP, pH 7-2, Zero-sodium solution pCa 9-6. 115 KCl, 20 NaCl, 14 EGTA, 1 CaCl2, 10 HEPES, 10 Mg-ATP, High-sodium solution pH 7-2, pCa 9-6.
correction for leakage current (Fig. 1B, 0). The transient outward current was derived by subtracting from the total outward current, the maintained current at the end of a 160 ms test pulse (Fig. 1B, A). The maintained component of outward current was defined as the current remaining at the end of a 160 ms test pulse, following leak subtraction (Fig. 1B, 0). It was apparent from these I-V revelations that the activation ranges of the transient and maintained currents were different. Furthermore, evaluation of the I-V relation of the transient current suggested the possible existence of two components of Ito, one showing a bell-shaped voltage dependence with a maximum around -20 mV, and the other with a monotonic voltage dependence in the range + 10 to + 60 mV (Fig. 1B, A/). Consistent with this idea, the time course of the inactivation process was much faster at voltages between -40 and -10 mV (average time constant, 18 ms (n = 3)) compared to more positive potentials (-10 to + 60 mV) (average time constant, 48 ms (n = 3)) in a manner similar to that seen in Purkinje fibres (Coraboeuf & Carmeliet, 1982). To study separately the characteristics of Imaintained and Ito, we investigated the effects of alterations of the holding potential on the two current components. In the holding potential range between -100 and -80 mV little change in It. occurred (compare 0 versus * in Fig. 2D). At holding potentials of -60 mV (Fig. 2B) and -50 mV, however, the transient outward current at +60 mV was reduced by 30 and 70 % (n = 5), respectively, compared to that recorded from the -80 mV holding potential (Fig. 2A). This was reflected in the I-V relation as a decrease in both the
TRANSIENT OUTWARD CURRENT IN RAT MYOCYTES
399
bell-shaped and linear components of It. (Fig. 2D, A and A). At a holding potential of -40 mV, the transient outward current appeared to be completely suppressed revealing the presence of a prominent calcium current (Fig. 2 C). Examination of the I-V relation at this holding potential showed that the bell-shaped region of Ito
A
~~+60 mv
\
*109~~ B
20
m
60
5+
/ 0
4/
0
/(nA) 3
o 0
A
A
A
o/
2-
0 20 60 40 V (mV) Fig. 1. Features of the peak (It.) and maintained ('maintained) components of outward current. A, outward current recorded from a single rat ventricular myocyte from a holding potential of -80 mV, activated in response to depolarizing steps delivered in 10 mV increments up to + 60 mV. The pulse to -70 mV activates no time-dependent current, the pulses to -60 and -50 mV activate only sodium current, while pulses above -50 mV activate increasing amounts of a transient outward current (Ito). At potentials positive to -10 mV a maintained outward current which shows no decay was activated. No net inward calcium current ICa was evident in these current records. Currents were both leak and capacitance subtracted. B, the current-voltage relation for the outward current expressed in three alternative ways: total outward current, following leak subtraction (0), outward current remaining at the end of a 160 ms depolarizing pulse (Imaintained), following leak subtraction (@) and transient outward current (Ito) obtained by subtracting the total and maintained outward currents (A). -60
-40
-20
occurred over the same voltage range as the negative bell-shaped curve of ICa (compare A and E, Fig. 2D). The maintained outward current showed a similar voltage dependence to the transient current as it was unaffected by shifts of holding potentials from -100 to
I. D. DUKES AND M. MORAD
400
-80 mV, but was significantly suppressed at a holding potential of -40 mV (n = 5, data not shown). Thus changes in the holding potential failed to separate either the different components of It. or the transient from the maintained outward current. The biphasic nature of the I-V relation of Ito may result either from two populations of channels activating over separate voltage ranges or alternatively that 500 pA
A ,
