J Physiol Volume 510, Number 3, 755-771, August 1, 1998
The Journal of Physiology (1998), 510.3, pp. 755-771
© Copyright 1998 The Physiological Society
A repolarization-induced transient increase in the outward current of the inward rectifier K+ channel in guinea-pig cardiac myocytes
Keiko Ishihara and Tsuguhisa Ehara
Department of Physiology, Saga Medical School, Saga 849-8501, Japan
Received 10 October 1997; accepted after revision 20 April 1998.
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ABSTRACT |
- Outward currents of the inwardly rectifying K+ current (IKir) in guinea-pig ventricular myocytes were studied in the presence of 1 mM intracellular free Mg2+ using the whole-cell patch-clamp technique.
- During repolarizing voltage steps following a large depolarizing pulse (> 0 mV), outward IKir increased transiently at voltages positive to the K+ equilibrium potential (EK, -84 mV for 5·4 mM extracellular [K+]). The rising phase was almost instantaneous, while the decay was exponential. The decay rate was faster at voltages closer to EK (time constants, 33·9 ± 9·8 and 4·8 ± 1·4 ms at -30 and -50 mV, respectively).
- The transient outward IKir was absent when the preceding depolarization was applied from -40 mV. Larger transient currents developed as the voltage before the depolarization was shifted to more hyperpolarized levels.
- Shift of the depolarizing voltage from > 0 mV to more negative ranges diminished the amplitudes of transient outward IKir and instantaneous inward IKir during the subsequent repolarizing steps positive and negative to EK, respectively. Since blockage of IKir by internal Mg2+ occurs upon large depolarization, and the block is instantaneously relieved at voltages negative to EK, the rising phase of the transient outward IKir was attributed to the relief of Mg2+ block at voltages positive to EK. Transient outward IKir was absent when intracellular [Mg2+] was reduced to 10 µM or lower.
- Prolongation of the repolarizing voltage step increased the amplitude of time-dependent inward IKir during the subsequent hyperpolarization, indicating the progress of a gating process (presumably the channel block by intracellular polyamine) during the decaying phase of outward IKir.
- Progressive prolongation of the depolarizing pulse (> 0 mV) from 100 to 460 ms decreased the transient outward IKir amplitude during the subsequent repolarizing step due to slow progress of the gating (polyamine block) at > 0 mV.
- Current-voltage relations measured using repolarizing ramp pulses (-3·4 mV ms-1) showed an outward hump at around -50 mV, the magnitude of which increased as the voltage before the conditioning depolarization (10 mV) was shifted to more negative levels. With slower ramp speeds (-1·5 and -0·6 mV ms-1), the hump was depressed at voltages near EK.
- Our study suggests that the relief of Mg2+ block may increase outward IKir during repolarization of cardiac action potentials, and that the resting potential, the level/duration of action potential plateau and the speed of repolarization influence the outward IKir amplitude.
- A kinetic model incorporating a competition between polyamine block and Mg2+ block was able to account for the time dependence of outward IKir.
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INTRODUCTION |
The inwardly rectifying K+ current (IKir), historically termed IK1 in the heart, maintains the resting membrane potentials of ventricular myocytes and Purkinje fibres near the equilibrium potential for K+ (EK). During the action potentials, IKir in these cells flows outwardly only in the voltage range near EK due to its strong inward rectification. This flow of outward IKir, however, is important for the final repolarization of the action potentials (Noble, 1975; Giles & Imaizumi, 1988; Carmeliet, 1993). The reduction of outward IKir caused by low external [K+] or external Cs+ block has been shown to result in a retardation of normal repolarization (early after-depolarization), which in turn induces ventricular tachiarrythmias (Cranefield & Aronson, 1988).
The channel gating causing the strong rectification of IKir (Carmeliet, 1982; Kurachi, 1985; Tourneur, Mitra, Morad & Rougier, 1987; Ishihara, Mitsuiye, Noma & Takano, 1989; Silver & DeCoursey, 1990; Stanfield et al. 1994) is now accounted for by the voltage-dependent blockage of the channel by intracellular polyamines (spermine/spermidine) (Lopatin, Makhina & Nichols, 1994; Ficker, Taglialatela, Wible, Henley & Brown, 1994; Lopatin, Makhina & Nichols, 1995; Ishihara, Hiraoka & Ochi, 1996; Lopatin & Nichols, 1996). Cytoplasmic Mg2+ has also been shown to block the outward current of the inwardly rectifying K+ (KIR) channels (Matsuda, Saigusa & Irisawa, 1987; Vandenberg, 1987). Under physiological conditions, it has been suggested that blockage of the channel by Mg2+ occurs at depolarized voltage levels, while spermine block takes place in the voltage range near EK (Ishihara et al. 1989, 1996). The role of the Mg2+ block, however, is unclear since the channel can exhibit a strong inward rectification in the absence of Mg2+ (Ishihara et al. 1989; Silver & DeCoursey, 1990; Stanfield et al. 1994).
By studying the cloned KIR channel IRK1 (Kir2.1), we recently found that the fast relief of Mg2+ block increases the outward current of IKir flowing during membrane repolarization (Ishihara, 1997). Since repolarization of cardiac action potentials is caused by a small net outward current (Cranefield & Aronson, 1988), we studied whether or not the same current component flows in cardiac myocytes. We show here that the outward current of native cardiac IKir transiently increases upon repolarization due to the relief of Mg2+ block and the subsequent gating process presumably caused by the polyamine block. Observations on this current component suggest that alterations of outward IKir amplitude modulate the repolarization phase of the cardiac action potentials under physiological and pathological conditions.
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METHODS |
Cell preparation
The procedure of isolating single ventricular cells from the adult guinea-pig heart was essentially the same as that used in previous studies (Powell, Terrar & Twist, 1980; Isenberg & Klöckner, 1982). Briefly, guinea-pigs (250-400 g body weight) were killed by an overdose of an intraperitoneal injection of sodium pentobarbitone (120 mg kg-1). The heart was excised, and was perfused at 37°C with Ca2+-free Tyrode solution containing 0·6 mg ml-1 collagenase (Wako, Osaka, Japan) using a Langendorff-type apparatus. After the enzyme treatment, the cells were dissociated in Kraft-Brühe (KB) solution (Isenberg & Klöckner, 1982), and then stored in the same solution at 4°C for later use. All procedures using animals were approved by the ethical committee of this institute (Saga Medical School).
Solutions
The Ca2+-free Tyrode solution contained (mM): 140 NaCl, 5·4 KCl, 0·5 MgCl2, 0·33 NaH2PO4, 5·5 glucose and 5 Hepes (titrated to pH 7·4 by NaOH). The KB solution contained (mM): 70 potassium glutamate, 30 KCl, 10 KH2PO4, 1 MgCl2, 20 taurine, 0·3 EGTA, 10 glucose and 10 Hepes (pH 7·2 with KOH). The standard external solution used for the voltage-clamp experiments contained (mM): 144·6 NaCl, 5·4 KCl, 1·8 CaCl2, 0·33 NaH2PO4 and 5 Hepes (pH 7·4 with NaOH). Unless otherwise noted, currents were recorded at 5·4 mM extracellular [K+]. When external [K+] was varied, equimolar NaCl was either supplemented or removed. Nicardipine (2 µM; Sigma), E-4031 (5 µM; a gift from Eisai, Tokyo, Japan) and ouabain (20 µM; Sigma) were always added in the external solutions to suppress the L-type Ca2+ current, the rapidly activating delayed rectifier K+ current (IKr), and the Na+-K+ pump current, respectively. The pipette solutions contained (mM): 30 KCl, 85 potassium aspartate, 10 KH2PO4, 5 or 2 EDTA, 1·9 K2ATP and 5 Hepes (pH 7·2 with KOH). Except for the experiment of Fig. 5B, the concentration of intracellular free Mg2+ was set at approximately 1 mM by adding 7·9 or 4·8 mM MgCl2 to the pipette solutions containing either 5 or 2 mM EDTA, respectively (Fabiato & Fabiato, 1979). The total [K+] in the pipette solutions was approximately 150 mM. All recorded membrane potentials were numerically corrected by -10 mV for the liquid junction potential between the pipette solution and the external solution.
Recording techniques and data analysis
Membrane currents were measured by the whole-cell patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) using an EPC-7 amplifier (List, Darmstadt, Germany). Patch pipettes were pulled from a Pyrex glass tube (1·5 mm o.d., 1·0 mm i.d.; Narishige, Tokyo, Japan) on a horizontal puller (Sutter Instruments Co., Novato, CA, USA). The resistance of the pipettes was typically 1·5-2·5 M
when filled with pipette solutions. Voltage stimulation and data acquisition were performed using the pCLAMP software (version 6, Axon Instruments) on a 486 IBM-PC clone through a Digidata 1200A A/D converter (Axon Instruments). All experiments were conducted at room temperature (20-23°C).
Currents were analysed using the pCLAMP software and KaleidaGraph (version 3, Synergy Software, Reading, PA, USA). The statistical values were given as the mean ± S.D. The values of the predicted EK were calculated by the Nernst equation using 150 mM internal [K+].
Computer simulation
Computation of the model was performed using QuickBASIC (Microsoft) on the 486 IBM-PC clone. The Euler iteration technique was used to solve the first-order differential equations describing the kinetic model.
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RESULTS |
Transient increase in outward IKir
The currents shown in Fig. 1A were recorded from a guinea-pig ventricular myocyte bathed in the standard extracellular solution with 5·4 mM K+, supplemented with nicardipine, E-4031 and ouabain to suppress the L-type Ca2+ current, IKr and the Na+-K+ pump current, respectively. The pipette solution contained approximately 1 mM free Mg2+. The current shown on the left was obtained using a 100 ms voltage step to 48 mV applied from a holding potential of -40 mV. Although such a depolarizing pulse is often used to activate the delayed rectifier K+ current (IK), time-dependent changes were not observed in the small outward current except for the capacitive transients, since the activation of IK is slow and small at room temperature (Kiyosue, Arita, Muramatsu, Spindler & Noble, 1993). In the right panel of Fig. 1A, a short hyperpolarizing voltage step to -118 mV was added to activate inward IKir before the depolarizing pulse to 48 mV. A small outward current component declined slowly during the depolarizing pulse. Furthermore, when the voltage was clamped back to -40 mV, the outward current increased transiently before reaching a steady level.
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Figure 1. Transient increase in outward IKir in the presence of 1 mM intracellular Mg2+
The currents in each column were recorded using the pulse protocol shown at the top. The external solutions contained 5·4 mM [K+] (A) and 0 mM [K+] (B). Difference currents (5·4 mM [K+] - 0 mM [K+]) are shown in C. Horizontal dashed lines superimposed on the pulse protocol and the current traces indicate the predicted EK (-84 mV) and the zero-current level, respectively.
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Removal of extracellular K+ has been shown to eliminate IKir (Ohmori, 1978), but not IK (Fan & Hiraoka, 1991). When the cell was exposed to the K+-free external solution (Fig. 1B), both the transient and sustained outward currents at -40 mV disappeared together with the large inward current at -118 mV and the small time-dependent outward current at 48 mV, suggesting that all of these currents were IKir. Figure 1C shows IKir isolated as the difference between the currents recorded at 5·4 and 0 mM external K+. The amplitude of outward IKir was small at the end of the depolarizing pulse, regardless of whether IKir was activated before the pulse or not. With the activation of IKir before depolarization, however, the amplitude of instantaneous outward IKir elicited on repolarization to -40 mV was increased.
The transient current was also blocked by external Ba2+ at a concentration that has been shown to block the outward current of cardiac IKir (Imoto, Ehara & Matsuura, 1987; Hirano & Hiraoka, 1988; Fig. 2). In the presence of 5 mM Ba2+, the inward current in the hyperpolarizing step (-119 mV) decreased time dependently, showing the characteristics of the block of inward IKir by Ba2+, and the transient and sustained outward currents at -40 mV disappeared.
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Figure 2. Block of transient IKir by external Ba2+
The currents were recorded under control conditions and in the presence of 5 mM Ba2+ using the pulse protocol shown at the top. Horizontal dashed lines superimposed on the pulse protocol and the current traces indicate the predicted EK (-84 mV) and the zero-current level, respectively.
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Current-voltage (I-V) relation of IKir during repolarizing voltage steps
The membrane currents shown in Fig. 3A were recorded at various voltages after the membrane was depolarized following a short hyperpolarizing step. A time-dependent change was noticed in the outward currents at various voltage levels. I-V relations of these currents are illustrated in Fig. 3B. The peak levels of the outward currents showed an inward rectification that was less strong than the rectification of sustained currents. The reversal potential (Erev) of the currents was close to the predicted EK (-84 mV at 20°C), and the currents were completely suppressed in the K+-free external solution (Fig. 3B). It was thus suggested that the currents observed during the repolarizing voltage steps were mostly IKir.
When external [K+] was varied, Erev of the currents shifted along with the shift of the predicted EK (-99 and -68 mV at 3 and 10 mM external [K+], respectively), and transient components were observed in the outward current region (Fig. 3C). As external [K+] was increased, the amplitude of transient currents (difference between the peak and steady-state levels of the outward currents) increased with that of sustained outward currents, which is compatible with the external [K+] dependence of the conductance of IKir. Since a prominent negative slope region was present in the peak outward I-V relations, the relations obtained at different [K+] produced a clear crossover, which is known to be a characteristic feature of IKir (Noble, 1975).
Time and voltage dependence of the transient current component
In the experiment of Fig. 3A, transient components were absent in the outward currents when the hyperpolarizing step (-118 mV) preceding the depolarizing pulses was omitted from the pulse protocol (currents not shown, but see Fig. 1A). To further characterize the transient currents, currents recorded using the protocol without the hyperpolarizing step were digitally subtracted from those shown in Fig. 3A. By using this method, the peak of the currents was observed more clearly, since the capacitive transient obscuring the current onset was removed (Fig. 4A). As the voltage was shifted to more negative levels (closer to EK), the decay of the current became faster. The time course of the decaying phase could be approximated using a single exponential function as shown by the curves superimposed on the current traces. The voltage dependence of the time constant was similar among different experiments (Fig. 4B). The mean values of the time constants were 33·9 ± 9·8 and 4·8 ± 1·4 ms (n = 10) at -30 and -50 mV, respectively.
Figure 4C shows the amplitude of the transient current components measured using the subtracting procedure. The transient current was observed at potentials negative to -10 mV, and its amplitude was maximal at around -50 to -60 mV. The current amplitudes normalized by the total cell capacitance varied among different experiments. This variation was roughly parallel to that observed in the density of inward IKir (data not shown). The mean values of the current amplitudes were 1·2 ± 0·5 and 3·1 ± 1·3 pA pF-1 (n = 7) at -30 and -50 mV, respectively.
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Figure 4. Transient component of outward IKir elicited in repolarizing voltage steps
A, transient current components were isolated as the difference between currents observed in repolarizing voltage steps in the presence and absence of the preceding hyperpolarizing step. Voltage steps were applied at the time indicated by the vertical line. The voltage for the step is indicated at the left of each trace. Horizontal dashed lines are the zero-current level. Decaying phases of the currents are superimposed with single exponential curves. The time constant is depicted above each trace. The experiment is the same as that shown in Fig. 3A. B, voltage dependence of the time constant of decaying phase. Different symbols indicate different experiments (n = 10). C, amplitude of transient current components. Current amplitudes were normalized to cell capacitance. Different symbols indicate different experiments (n = 7).
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Relief of the block of IKir by internal Mg2+ increases outward IKir
We next examined the mechanism of the transient increase in outward IKir. When the voltage level preceding the depolarizing pulse was shifted to a more hyperpolarized level, a larger transient outward IKir developed during the subsequent repolarization to -40 mV (Fig. 5Aa-c). This observation suggests that the opening of KIR channels in advance of depolarization changed the state of the channels during the depolarization and the succeeding repolarization. Figure 5Ad shows the relation between the voltage of the pre-pulse and the amplitude of the transient outward IKir. The membrane conductance in the pre-pulse, which is mostly attributable to the conductance of IKir, is also plotted in the graph as a function of the membrane voltage. The amplitude of transient IKir is observed to correlate with IKir conductance in the voltage preceding the depolarizing pulse. When the depolarizing pulse was applied from voltages negative to -110 mV, where IKir conductance was maximal, the amplitude of transient IKir reached its highest level. When the voltage prior to the depolarizing pulse was set at -90 or -80 mV near the predicted EK, the amplitude of transient IKir was 53 ± 8 % (n = 5) or 33 ± 9 % (n = 3) of the maximum, respectively.
By shifting the pre-pulse to hyperpolarized levels, inward IKir observed on hyperpolarization after the depolarizing pulse also changed (Fig. 5Aa-c); the amplitude of IKir which showed a rapid exponential increase diminished, while that of IKir which flowed at the instance of hyperpolarization increased (arrow). When the membrane is depolarized from a hyperpolarized level, internal free Mg2+ increases the amplitude of instantaneous inward IKir during the subsequent hyperpolarization in a dose-dependent manner (Ishihara et al. 1989). This phenomenon implies that Mg2+ ions block the opened KIR channels upon depolarization, and that the relief of the block at hyperpolarized levels is fast enough to cause an instantaneous increase in IKir. Thus, the currents illustrated in Fig. 5A suggest that the fraction of KIR channels being blocked by Mg2+ during depolarization increased by hyperpolarizing the membrane before the depolarization, and that the relief of Mg2+ block also increased the instantaneous outward IKir at the following voltage of -40 mV. Consistent with this idea, no transient outward IKir was observed when intracellular free [Mg2+] was reduced to 10, 1 or below 0·01 µM (n = 21) (Fig. 5B).
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Figure 5. Evidence for the involvement of Mg2+ block of IKir in the occurrence of transient outward IKir
Aa-c, influence of the voltage in the pulse preceding the depolarizing pulse (48 mV, 100 ms) on IKir during the subsequent repolarizing steps at -40 and -118 mV. An increase in the instantaneous component of inward IKir at -118 mV is designated by arrows. Ad, relation between the voltage in the pre-pulse and the amplitude of transient outward IKir at -40 mV. Amplitudes of transient current components were measured using the subtracting procedure (Fig. 4), and were normalized with the maximum amplitude observed by setting the voltage before depolarizing pulse at ~-120 mV. Different symbols indicate different experiments (n = 6). Relation between voltage and chord conductance (G = I/(V - Erev)), obtained using 20 ms voltage steps (in 10 mV increments) applied from a holding potential of -40 mV, is also shown by the dotted curve (vertical line, Erev = -82 mV). The given conductance is mostly applicable to IKir in the absence of the L-type Ca2+ current. Values of chord conductance were normalized with the maximum conductance observed at -118 mV (105 nS). The conductance and the currents shown in a-c were obtained from the same experiment. B, currents recorded using the pipette solution containing approximately 10 µM free Mg2+. Note that transient increase was absent in the outward current at -40 mV. In A and B, horizontal dashed lines superimposed on the pulse protocol and the current traces indicate the predicted EK (-84 mV) and the zero-current level, respectively.
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When the voltage in the depolarizing pulse was shifted closer to EK, the amplitude of instantaneous inward IKir decreased and that of time-dependent inward IKir increased during the following hyperpolarization (Fig. 6A). This phenomenon indicates that the fraction of IKir being blocked by Mg2+ decreased at voltages near EK (Ishihara et al. 1989). Figure 6B shows that the amplitude of transient outward currents at -40 mV also decreased by decreasing the preceding depolarization. The relation between the voltage during the depolarizing pulse and the amplitudes of the instantaneous currents at the subsequent voltages of -40 and -119 mV (Fig. 6C) indicates that the amplitudes of the instantaneous currents at -119 and -40 mV closely correlated. This observation supports the view that the transient outward IKir and the instantaneous inward IKir were caused by a common mechanism, namely the relief of Mg2+ block.
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Figure 6. Correlation between the amplitudes of instantaneous inward IKir and transient outward IKir
A, influence of the depolarizing voltage on inward IKir during the subsequent hyperpolarization to -119 mV. The membrane was briefly hyperpolarized from -40 mV to activate IKir before depolarizing to -30, -10 and 29 mV for 100 ms. In an attempt to evaluate the instantaneous current amplitude on hyperpolarization, exponential curves (time constant, 1·8 ms) fitted to time-dependent phases were extrapolated to time zero. The time zero was arbitrarily set at a time when capacitive transients decayed to e-2 of its maximum amplitude. B, influence of the depolarizing voltage on outward IKir during the subsequent repolarization to -40 mV. Depolarizing pulses were the same as those used in A. To measure the peak outward current amplitudes for the plot shown in C, transient current amplitudes were measured using the subtracting procedure (Fig. 4), and were summed up with sustained-current amplitudes. In A and B, horizontal dashed lines superimposed on the pulse protocol and the current traces indicate the predicted EK (-84 mV) and the zero-current level, respectively. C, amplitudes of instantaneous inward IKir at -119 mV ( ) and peak outward IKir at -40 mV ( ), plotted against the voltage in the preceding depolarizing pulse. Amplitudes of the outward currents at the end of the depolarizing pulses are also plotted ( ) to denote that the instantaneous currents at -119 and -40 mV are not attributable to the IKir conductance in the depolarizing pulses.
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Gating of IKir during the decaying phase of outward IKir
Studies on cloned KIR channels with strong inward rectification (Kir2.0 subfamily) have suggested that the time-dependent increase of inward IKir on hyperpolarization reflects the relief of the block of the KIR channel by internal polyamines (spermine/spermidine) (Lopatin et al. 1994; Ficker et al. 1994). The currents recorded using the envelope test (Fig. 7A) revealed that the channel gating, which is now accounted for by the polyamine block of the channel, proceeds during the decaying phase of the transient outward currents. When the repolarizing voltage step at -30 or -50 mV was progressively prolonged from 1 to 150 ms, the amplitude of instantaneous inward IKir decreased, while that of time-dependent inward IKir increased, during the subsequent hyperpolarizing step. The time course of the envelope became faster as the voltage in the repolarizing step became more negative, closer to EK; the time constants were 36, 14 and 8 ms at -30, -40 and -50 mV, respectively (Fig. 7B). These values were similar to the time constants obtained for the decaying phase of the outward currents (35, 10 and 5 ms at the corresponding voltages in this experiment; see also Fig. 4B), indicating the involvement of the gating process (polyamine block) in the decaying phase of outward IKir.
Prolongation of depolarization decreases the peak amplitude of outward IKir during the subsequent repolarization
In the presence of intracellular Mg2+, the gating process (polyamine block) of IKir proceeds at a slower rate as the membrane potential becomes more positive (Fig. 8A; see also Ishihara et al. 1989, 1996). The gradual redistribution from the blocked state by Mg2+ to the 'closed state' (the blocked state by polyamine) of the channel has been proposed to underlie the time course of this phenomenon. Upon depolarization to voltages positive to 0 mV, which are close to the levels of the action potential plateau in ventricular myocytes, the time course of this process was so slow that it was not completed within 500 ms (Fig. 8A and B). It appeared to proceed for more than 1 or 2 s, but the entire time course could not be measured using long depolarizing pulses because the activation of IK hampered the analyses. As expected from the above, progressive prolongation of depolarizing pulses from 100 to 460 ms gradually decreased the peak amplitude of the transient outward IKir during the subsequent repolarization (Fig. 9). This reduction of transient IKir occurred faster at 10 mV than at 50 mV, which correlated with the observation that the progress of the gating process (polyamine block) was faster at 10 mV than at 50 mV (Fig. 8B).
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Figure 9. Prolongation of depolarization diminishes transient outward IKir during the following repolarization
Transient outward IKir observed following various durations (100, 220, 340, 460 ms) of depolarization is shown. The voltages in depolarizing pulses were 10 mV (left) and 50 mV (right). Horizontal dashed lines superimposed on the pulse protocol and the current traces indicate the predicted EK (-84 mV) and the zero-current level, respectively. Current levels in the hyperpolarizing step to -119 mV are clipped. Decreases in the peak amplitude of outward IKir at -50 mV are designated by the dotted lines.
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Outward IKir during repolarizing ramp pulses
To examine the physiological role of the outward IKir component generated by the relief of Mg2+ block in the repolarization phase of the action potential, we recorded currents using repolarizing ramp pulses. The ramp speed was set at -3·4 to -0·6 mV ms-1, which may correspond to the actual repolarizing rate in the action potentials of ventricular myocytes. In Fig. 10A, a conditioning depolarizing step was applied directly from -40 mV, or following a pre-pulse to -90 or -120 mV, and then a repolarizing ramp (-3·4 mV ms-1) was applied. As expected from the results of the experiments performed using the repolarizing step pulses (Fig. 5), the more negative the voltage prior to the depolarization was, the larger the outward hump in the I-V relation became, indicating the flow of the 'transient component' of outward IKir during the ramp pulse. As the transient increase in outward IKir was not observed during repolarizing steps when the preceding depolarization was applied from -40 mV, the current component generated by the relief of Mg2+ block during the repolarizing ramp was estimated by subtracting the current obtained without using the pre-pulse from those obtained using the pre-pulse (Fig. 10A, lower panel). The current amplitude at voltages negative to -50 mV was small, compared with the transient outward IKir amplitude measured using the step-pulse protocol (Fig. 4C). This is explained by the finding that the rate of the decay of the current becomes faster as the voltage becomes closer to EK (Fig. 4B). Therefore, when the ramp speed was slower (-1·5 and 0·6 mV ms-1), the outward hump was further depressed in the voltage range near EK (Fig. 10B).
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Figure 10. Outward IKir during a repolarizing ramp pulse
A, I-V relations of outward IKir measured using repolarizing ramp pulses applied from 10 to -160 mV in a 50 ms time period (-3·4 mV ms-1). Before the ramp pulse, a conditioning depolarizing step (10 mV, 100 ms) was applied directly from a holding potential of -40 mV ( ), or following a pre-pulse to -90 mV ( ) or -120 mV (). Lower panel, I-V relations of the outward IKir component generated by the relief of Mg2+ block during repolarizing ramp pulses. The current components were obtained by subtracting the current obtained without using the pre-pulse from those obtained using the pre-pulse. B, effects of the ramp speed on the amplitude of outward IKir. The conditioning step depolarization (10 mV, 100 ms) was applied following a pre-pulse to -120 mV, and then ramp pulses were applied from 10 to -140 mV over a 50 ms (), 100 ms () and 250 ms ( ) time period (-3·0, -1·5 and -0·6 mV ms-1, respectively). For comparison, the I-V relation of the current obtained without using the pre-pulse is also plotted (-3·0 mV ms-1, dashed line). Current levels during ramp pulses were numerically compensated for the capacitive current calculated by the cell capacitance multiplied by the ramp speed.
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DISCUSSION |
Transient outward currents observed in the presence of internal Mg2+ are IKir
The transient increase in outward IKir was first found in the presence of intracellular free Mg2+ (0·4-1·1 mM) when IKir was expressed by transfecting the IRK1 gene into L cells (a murine fibroblast cell line) (Ishihara, 1997). Since the electrophysiological properties of IRK1 IKir are close to those of native IKir in ventricular myocytes (Stanfield et al. 1994), we assumed that the same current change may be found for the native cardiac IKir. Transient outward currents were elicited in repolarizing voltage steps when intracellular free [Mg2+] was adjusted to approximately 1 mM by using either 2 or 5 mM EDTA, but not to 10 µM or lower concentrations (Fig. 5). The currents showed the characteristics of IKir, as they were blocked by external Ba2+ (Fig. 2), and disappeared under external K+ free conditions (Figs 1 and 3). The external [K+] dependence of the currents was also typical for IKir (Fig. 3). Among the ionic currents known in ventricular myocytes, the transient currents were similar to the tails of IKr because the outward current of IKr is small during depolarization (inward rectification), and increases transiently when the membrane potential is brought back into the negative range (Sanguinetti & Jurkiewicz, 1990). However, the currents shown in this study were all recorded in the presence of E-4031, which selectively blocks IKr (Sanguinetti & Jurkiewicz, 1990). Furthermore, the transient components of outward IKir were clearly distinguishable from the tails of IKr, since they were not observed when the conditioning depolarization was applied from a holding potential of -40 mV (Fig. 5), while IKr is known to be activated using a holding potential of -40 mV (Sanguinetti & Jurkiewicz, 1990).
Mechanisms underlying the transient increase in outward IKir
The transient increase in outward IKir at the repolarized voltage level represents the opening of a 'channel gate' followed by the closing of 'another gate'. In cardiac IKir, it has been shown that a time-dependent gating process that was constantly observed under extensive internal perfusion mainly causes the strong inward rectification of the current in the absence of intracellular Mg2+, while Mg2+ ions at physiological concentrations (0·5-1·2 mM in free form; Murphy, Freudenrich & Lieberman, 1991) block the channels and slow this gating at depolarized voltage levels (Ishihara et al. 1989). In the present study, we showed that the relief of this Mg2+ block increases the outward current, and the above gating subsequently reduces the current again at the repolarized levels positive to EK. Examination of macroscopic IKir in the inside-out patches has become possible by the expression of cloned KIR channels in the cells adequate for the macro-patch configuration. This revealed that the blockage of the channels by internal cationic polyamines (spermine/spermidine/putrescine) causes the inward rectification of IKir (Lopatin et al. 1994; Ficker et al. 1994; Fakler, Brändle, Glowatzki, Weidemann, Zenner & Ruppersberg, 1995). Although we have not yet succeeded in obtaining the same evidence in the native cardiac IKir, the above time-dependent gating of cardiac IKir is strikingly similar to that of IRK1 IKir, which has been recognized to represent the blockage of the channel by internal spermine (Ishihara et al. 1996; Ishihara, 1997).
Problems in methodology
Polyamines and Mg2+ have been shown to block KIR channels with different kinetics (Lopatin et al. 1995; Yang, Jan & Jan, 1995; Ishihara et al. 1996; Lopatin & Nichols, 1996). The rate of the relief of the block at hyperpolarized voltage levels decreases with increasing charge on the particles: the increase in inward IKir due to the relief of Mg2+ and putrescine(2+) block is virtually instantaneous, while those caused by the relief of spermidine(3+) and spermine(4+) block are time dependent. It is thus possible to estimate distribution of the different blocked states by analysing the time course of the inward current observed upon hyperpolarization. For this purpose, it would be essential to record currents under a fast voltage clamp, because the time constants of the time-dependent phase of inward IKir, which is usually fitted by a single exponential function, are as small as several milliseconds in ventricular cells. However, since the methods allowing a fast clamp generally involve extensive intracellular perfusion which may significantly decrease intracellular polyamines, we used the tight-seal whole-cell clamp method despite its disadvantage of a slow voltage clamp. Using this method, alterations of IRK1 IKir in L cells, which could be explained by a decrease in intracellular spermine level, still occurred slowly during experiments (Ishihara et al. 1996; Ishihara, 1997). In cardiac IKir, however, no such changes were evident. This may be due to differences in the size and morphology between the cells. As noted above, the high series resistance and large membrane capacitance of ventricular cells (123 ± 42 pF, n = 20) gave rise to slow capacitive currents (time constants, 0·69 ± 0·3 ms; n = 20), which significantly overlapped with the time-dependent phase of inward IKir (e.g. Fig. 6A). We were thus limited to evaluating only the changes in the amplitudes of time-dependent and instantaneous components of inward IKir. Since the relief of spermidine block is faster than that of spermine block, the component reflecting the spermidine unblock may be obscured under capacitive transients in the present study. We previously showed in the study of IRK1 that the effects of intracellular putrescine on IKir are similar to those of intracellular Mg2+ (Ishihara, 1997). Thus the instantaneous component of inward IKir may also include the component reflecting the relief of putrescine block. However, the low level of endogenous putrescine in mammalian cells (Suzuki, He, Kashiwagi, Murakami, Hayashi & Igarashi, 1994) implies that putrescine block may be a minor component in cardiac IKir.
A kinetic model simulating the transient increase in outward IKir
To understand the molecular mechanisms underlying the transient increase in outward IKir, we first tried to simulate the current by a kinetic model with only one 'spermine-block' state (Bspm), one open state (O) and one 'Mg2+-block' state (BMg):
Model 1
, 
, µ and 
are the rate constants of the state transitions, which all depend on the voltage. In this model, the fraction of IKir being blocked by Mg2+ (FMgBlock) in the steady state is:
FMgBlock = KMg/(1 + KMg + Kspm),
where Kspm is / and KMg is µ/. At positive voltages where Kspm >> 1:
FMgBlock 
1/(1 + (Kspm/KMg)).
This formula indicates that the ratio Kspm/KMg determines the state distribution. In order to simulate the state transition BMg 
O Bspm upon repolarizing voltage change, the value of Kspm/KMg is expected to be small at depolarized levels, and then to become larger at voltages near EK.
The rate constants shown in the upper panel of Fig. 11A were defined to reproduce the steep inward rectification of IKir at around EK caused by the gating kinetics presumably reflecting the kinetics of spermine block, and the relatively mild rectification caused by the Mg2+ block (middle panel) (Kurachi, 1985; Tourneur et al. 1987; Matsuda, 1988; Silver & DeCoursey, 1990; Lopatin et al. 1994; Yang et al. 1995). The rate constants and reconstitute the instantaneous BMg O and the exponential time course of Bspm O observed in inward IKir at voltages negative to EK, respectively (Ishihara et al. 1989). The value of was obtained from the time course of outward IRK1 IKir measured in the presence of 1 µM internal spermine (Ishihara, 1997). The value of µ at 1 mM Mg2+ was arbitrarily defined by referring to the studies of IRK1 IKir (Lopatin et al. 1994; Yang et al. 1995). With these rate constants, the value of Kspm/KMg increases as the voltage becomes more positive, because the voltage dependence of Kspm is steeper than that of KMg (Fig. 11A, lower panel). It is thus difficult to reproduce the increase in FMgBlock at positive voltages by using model 1. Instead, we found that the following kinetic model used in previous works (Ishihara et al. 1989; Oliva, Cohen & Pennefather, 1990) simulates the findings nicely:
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Figure 11. A model simulating cardiac IKir
A, upper panel: blocking and unblocking rates for spermine block (dashed lines) and Mg2+ block (continuous lines) used in the calculations were:
(S-1) = 200 exp[-0·035 (V - EK)]/{exp[0·06 (V - EK)] + 1},
(S-1) = 300 exp[0·09 (V - EK)]/{exp[0·075 (V - EK - 37)] + 1},
µ (S-1) = 600 exp[0·032 (V - EK)]/{exp[0·09 (V - EK - 140)] + 1},
(S-1) = 2800 exp[-0·03(V - EK)] + 170,
where V and EK are in millivolts. and µ are assumed to be the values at 1 µM [spermine] and 1 mM [Mg2+], respectively. Middle panel: voltage-dependent decrease in FOpen due to spermine block alone (calculated by 1/(1 + Kspm), dotted line) or Mg2+ block alone (calculated by 1/(1 + KMg) in both models 1 and 2, continuous line). Lower panel: Kspm, KMg and (KMg)3 were calculated from the rate constants. B, steady-state FOpen (dotted line), FSpBlock (dashed line) and FMgBlock (continuous line) calculated by model 2. FOpen was calculated by (1 + KMg)2/{(1 + KMg)3 + Kspm}, FSpBlock by Kspm/{(1 + KMg)3 + Kspm} and FMgBlock by KMg (1 + KMg)2/{(1 + KMg)3 + Kspm}. C, calculation of FOpen, FSpBlock and FMgBlock during 500 ms step depolarizations to -30 and 10 mV applied from -120 mV (EK = -80 mV). FOpen was calculated by PO + 2/3PMg1 + 1/3PMg2, FSpBlock by Pspm, and FMgBlock by 1/3PMg1 + 2/3PMg2 + PMg3.
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Model 2
In this model, one channel is blocked by up to three Mg2+ ions, and the channel blocked by one (OMg1) or two (OMg2) Mg2+ ions shows two-thirds or one-third of the unit conductance, respectively, while that blocked by three Mg2+ ions (BMg3) becomes non-conductive. IKir is thus proportional to PO + 2/3PMg1 + 1/3PMg2, where Pspm, PO, PMg1, PMg2 and PMg3 represent the probabilities of the channel in state Bspm, O, OMg1, OMg2 and BMg3, respectively (Pspm + PO + PMg1 + PMg2 + PMg3 = 1). This model was originally developed in the study of the cardiac KIR channel, which analysed the subconductance levels of single channel currents induced by cytoplasmic Mg2+ (Matsuda, 1988). Similar subconductance levels have also been shown for IRK1 channel current (Omori, Oishi & Matsuda, 1997). Although there is thus far no direct evidence to support such a structure of the KIR channel, one channel may possess multiple Mg2+ binding sites that are apparently independent of each other (Root & MacKinnon, 1994).
In model 2, FMgBlock in the steady state is described as:
FMgBlock = 1/3PMg1 + 2/3PMg2 + PMg3
= KMg (1 + KMg)2/((1 + KMg)3 + Kspm).
At positive voltages where KMg >> 1:
FMgBlock 1/(1 + Kspm/(KMg)3).
This formula indicates that the ratio Kspm/(KMg)3 determines the state distribution, and that the channels may be blocked by Mg2+ even if Kspm >> KMg. With the rate constants illustrated in Fig. 11A, the voltage dependence of the value (KMg)3 is steeper than that of Kspm (Fig. 11A, lower panel). Thus the value of Kspm/(KMg)3 becomes small at positive voltages, and FMgBlock increases accordingly (Fig. 11B) as has been shown in our experiments (Fig. 6A; Ishihara et al. 1989, 1996).
Model 2 can also simulate the slow time course of redistribution BMg O Bspm during a large depolarization. Figure 11C shows the result of the calculation of FOpen (proportional to IKir; calculated by PO + 2/3PMg1 + 1/3PMg2), FSpBlock (the fraction of IKir being blocked by spermine; the same as Pspm) and FMgBlock during 500 ms step depolarizations applied from a voltage negative to EK (-120 mV with EK = -80 mV). On depolarization to a voltage 90 mV positive to EK (10 mV with EK = -80 mV), FMgBlock was observed to gradually decrease until it finally reaches a small value in the steady state (Fig. 11B). This happens because the increases in PMg2 and PMg3 significantly lower the chance of O Bspm in model 2. The time course of the increase in FSpBlock effectively simulates the slow time course of the envelopes during a large depolarization observed in experiments (Fig. 8). Even by adopting a different combination of rate constants, this slow redistribution BMg O Bspm was not simulated using model 1.
As shown in Figs 12 and 13, the findings in the present study were satisfactorily simulated by using model 2. Figure 12 shows the calculation of FOpen, FSpBlock and FMgBlock during the repolarizing pulse protocol. On repolarization to voltages positive to EK following a large depolarizing step, FOpen rapidly increased with the decrease in FMgBlock. FOpen then gradually decreased with the increase in FSpBlock. The time course of the decay of FOpen was speeded up by shifting the voltage in the pulse closer to EK. This is because the shift in the main component of FOpen from PMg2 toward PMg1 and PO (Fig. 12, right panel) increased the chance of O Bspm. The time courses of FOpen and FSpBlock are similar to those of the decaying phase of outward IKir (Fig. 4) and the envelope of time-dependent inward IKir (Fig. 7) at the corresponding voltages, respectively. The results of calculations illustrated in Fig. 13 reproduce the features of Figs 5, 6 and 9. The model simulates well the reduction of the peak amplitude of transient outward IKir, which occurred with the elevation of the voltage preceding depolarization (Fig. 13A), with the prolongation of depolarization (Fig. 13B) and with the decrease in the level of depolarization (Fig. 13C), which were all caused by the decrease in FMgBlock during depolarization.
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Figure 12. Computer simulation of transient outward IKir during repolarizing voltage steps at different voltage levels
Left, FOpen (dotted line), FSpBlock (dashed line) and FMgBlock (continuous line) during the pulse protocol shown above each plot. FOpen was calculated by PO + 2/3PMg1 + 1/3PMg2, FSpBlock by Pspm, and FMgBlock by 1/3PMg1 + 2/3PMg2 + PMg3. The amplitude of IKir is proportional to FOpen. Calculations were performed using EK = -80 mV. Right, contribution of PMg2, PMg1 and PO to FOpen.
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Figure 13. Computer simulation of transient outward IKir in repolarizing voltage steps
A, simulation of the effect of the voltage (-40, -80 and -120 mV) preceding the step depolarization (50 mV, 100 ms) on the amplitude of transient outward IKir during the subsequent repolarization to -40 mV. B, simulation of the effect of duration (100 and 500 ms) of step depolarization (10 mV) on the amplitude of transient outward IKir during the subsequent repolarization to -40 mV. C, simulation of the effect of the level (-30, -10 and 30 mV) of step depolarization (100 ms, applied from -120 mV) on the amplitude of transient outward IKir during the subsequent repolarization to -40 mV. In A-C, FOpen (dotted line), FSpBlock (dashed line) and FMgBlock (continuous line) during the indicated voltage protocol are shown. FOpen was calculated by PO + 2/3PMg1 + 1/3PMg2, FSpBlock by Pspm, and FMgBlock by 1/3PMg1 + 2/3PMg2 + PMg3. The amplitude of IKir is proportional to FOpen. Calculations were performed using EK = -80 mV.
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Physiological relevance
The time dependence of the outward current of IKir is not well understood (Ibarra, Morley & Delmar, 1991; Shimoni, Clark & Giles, 1992). Our study indicates the presence of a novel time-dependent component of outward IKir that may contribute to repolarization of the cardiac action potentials. Since this current component rapidly decays at voltage levels near EK (Fig. 4), it may accelerate repolarization mainly at potentials positive to -50 mV under physiological conditions (Fig. 10). Our findings (Figs 5, 6 and 9) also suggest that the resting potential and the level and length of the plateau phase (phase 2) of the action potential influence the amplitude of outward IKir, and thereby modulate the phase 3 repolarization.
Under pathological conditions, it has been suggested that a decrease in the net outward current flowing during repolarization interrupts normal repolarization, leading to generation of a depolarizing after-potential (early after-depolarization) which causes ventricular arrhythmias (Cranefield & Aronson, 1988; January & Moscucci, 1992; Hiraoka, Sunami, Zheng & Sawanobori, 1992). The time-dependent feature of outward IKir can be a factor responsible for the generation of early after-depolarizations, since the slowing of repolarization may reduce this current component (Fig. 10), which further hinders repolarization. A resting potential positively deviating from EK (for example, due to low external [K+]) and a prolongation of the action-potential plateau may be arrythmogenic to some extent due to the reduction of outward IKir. Cell magnesium deficiency may also be disadvantageous for repolarization for the same reason. The actual contribution of IKir to the repolarization of the cardiac action potential has to be further examined in the presence of intracellular Mg2+.
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Acknowledgements
We thank Drs M. Hiraoka, A. Noma, S. Oiki, K. Ono, H. Matsuura and T. Shioya for useful discussions. We also thank Dr B. Quinn for reading the manuscript, and Miyuki Fuchigami for her secretarial assistance. This work was supported by the Grant-in-Aid for Scientific Research on Priority Areas of 'Channel-Transporter Correlation' from the Ministry of Education, Science and Culture of Japan, and by the Japan Heart Foundation and IBM Japan Research Grant.
Corresponding author
K. Ishihara: Department of Physiology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Email: keiko{at}post.saga-med.ac.jp
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Introduction
