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Am J Physiol Cell Physiol 279: C603-C610, 2000;
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Vol. 279, Issue 3, C603-C610, September 2000

Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca2+ channel currents

Sayaka Mitarai1,2, Muneshige Kaibara1, Katsusuke Yano2, and Kohtaro Taniyama1

1 Department of Pharmacology and 2 The Third Department of Internal Medicine, Nagasaki University, School of Medicine, Nagasaki 8528523, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the inactivation process of macroscopic cardiac L-type Ca2+ channel currents using the whole cell patch-clamp technique with Na+ as the current carrier. The inactivation process of the inward currents carried by Na+ through the channel consisted of two components >0 mV. The time constant of the faster inactivating component (30.6 ± 2.2 ms at 0 mV) decreased with depolarization, but the time constant of the slower inactivating component (489 ± 21 ms at 0 mV) was not significantly influenced by the membrane potential. The inactivation process in the presence of isoproterenol (100 nM) consisted of a single component (538 ± 60 ms at 0 mV). A protein kinase inhibitor, H-89, decreased the currents and attenuated the effects of isoproterenol. In the presence of cAMP (500 µM), the inactivation process consisted of a single slow component. We propose that the faster inactivating component represents a kinetic of the dephosphorylated or partially phosphorylated channel, and phosphorylation converts the kinetics into one with a different voltage dependency.

channel phosphorylation; whole cell patch clamp


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CALCIUM CHANNEL CURRENTS ACTIVATE with membrane depolarization and inactivate over time. The inactivating property has an important role in regulating intracellular Ca2+ concentration and action potential duration of cardiac cells. Inactivation of the currents is modulated by at least three factors: 1) membrane potential, 2) Ca2+, and 3) phosphorylation of the channel (19). Attempts have been made to clarify relations between the observed inactivation process of the currents and these three factors.

Inactivation of cardiac L-type Ca2+ channel currents is accelerated by Ca2+ passing through the channel (15, 17, 20) and by intracellular Ca2+ released from the sarocplasmic reticulum (25), hence the inactivation process is complicated. Although Ca2+ channel currents carried by cations other than Ca2+ show a relatively slow decline in the absence of Ca2+-mediated inactivation (9, 15, 17, 20), it has been reported that decay of these currents carried by Ba2+, Sr2+, or Na+ is not fitted by a single-exponential function (3, 6, 10, 15). Kass and Sanguinetti (15) reported that decay of the currents carried by Ba2+ or Sr2+ was best fitted by functions with a two-exponential process. They proposed that the observed data might be explained by two populations of Ca2+ channels with different inactivation kinetics.

Stimulation of the beta -receptor-cAMP cascade by isoproterenol and effects of isoproterenol on the cardiac L-type Ca2+ channel have been extensively studied (14). In single-channel studies, isoproterenol increases open-state probability by increasing duration of the available state (4, 21) and prolonging open time of the channel (27). These effects of phosphorylation on channel kinetics were also evident using the phosphatase inhibitor okadaic acid (23). Although it has been demonstrated that isoproterenol slows the decay of the outward current through the Ca2+ channel at high-membrane potential in frog ventricular heart cells (2), these changes in kinetics of single-channel currents have not been thoroughly explored in the case of inward whole cell currents.

It has been suggested that some populations of the Ca2+ channels in cardiac myocytes are phosphorylated without stimulation of cAMP production (14, 23). Consequently, in whole cell recordings in the absence of exogenous stimulation of the beta -receptor-cAMP cascade, the inactivation process of macroscopic currents may reflect two populations of Ca2+ channels, i.e., phosphorylated and dephosphorylated. We investigated the inactivation process of macroscopic Na+ currents through the cardiac L-type Ca2+ channel and the effects of isoproterenol on this inactivation process. We report here that two distinct inactivation processes are present in the case of the Na+ currents through the channel and that isoproterenol converts these two inactivation processes into a slow one. We also found that N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of cAMP-dependent protein kinase (5), attenuated the effects of isoproterenol.


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

Ca2+ containing solution for whole cell current recording contained (in mM) 144 NaCl, 0.33 NaH2PO4, 5.4 CsCl, 0.5 MgCl2, 1.8 CaCl2, 5.5 glucose, and 5 HEPES-NaOH buffer (pH 7.4). Ca2+-free solution was prepared by omitting CaCl2 from the Ca2+ solution and adding 0.1 mM EGTA. The Tyrode solution contained (in mM) 144 NaCl, 0.33 NaH2PO4, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 5.5 glucose, and 5 HEPES-NaOH buffer (pH 7.4). The pipette solution contained (in mM) 110 cesium aspartate, 20 CsCl, 2 MgCl2, 3 MgATP, 10 EGTA, 5 HEPES, 0.5 NaGTP, and 5 NaCl, the pH was adjusted to 7.1. In some experiments, 500 µM cAMP was added to the pipette solution. H-89 was stored in 3 vol% ethanol solution at a concentration of 10 mM at -30°C. Single ventricular cells were isolated from guinea pig hearts, using a modification of a reported method (13). In brief, guinea pigs weighing under 250 g were anesthetized with pentobarbital sodium, the dissected hearts were mounted on a Langendorff apparatus, and the hearts were perfused at 37°C first with the Tyrode solution and then with CaCl2-omitted Tyrode solution. Finally, the hearts were perfused with CaCl2-omitted Tyrode solution containing collagenase. Thereafter, single ventricular cells were preserved in storage solution (12).

Membrane currents from single ventricular cells were recorded using the whole cell patch-clamp method (7) with an EPC-7 patch-clamp amplifier. The glass microelectrodes we used had a tip resistance ranging from 1.5 to 2.5 MOmega . The liquid junction potential between the pipette solution and the Tyrode solution, ~-12 mV, was canceled in each experiment. K+ currents were blocked with intracellular and extracellular Cs+ substituted for K+. Na+ channel current and T-type Ca2+ channel current were inactivated by setting the holding potential at -40 mV. Other currents were subtracted using currents recorded in the presence of 2 µM nifedipine and 500 µM CdCl2. Series resistance was compensated up to 75%. Currents and voltage signals were analyzed using a personal computer. Inactivation time course of nifedipine/Cd2+-sensitive currents was fitted with exponential functions using least squares (Kaleida Graph, version 3.08, Synergy Software). Comparisons were made using a paired or unpaired Student's t-test where appropriate and one-way ANOVA complemented by Dunn's procedure as a multiple comparison procedure. All data are presented as means ± SE. All experiments were done at 35-36°C.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inactivation process of Na+ currents through the cardiac L-type Ca2+ channel. L-type Ca2+ channel currents were recorded in the whole cell voltage-clamp configuration at +10 mV from the holding potential of -40 mV at 5-s intervals in the presence of external Ca2+ and subsequently in the absence of external Ca2+. Peaks of the inward currents are consecutively plotted against time in Fig. 1A. Application of Ca2+-free solution resulted in a decrease of inward currents, then stable inward currents were observed ~3 min after the application. Stable inward currents are carried mainly by Na+ after extracellular Ca2+ has been decreased with EGTA (6, 9, 18). The inward currents were abolished by nifedipine (2 µM) and CdCl2 (500 µM). Subtracted current traces, i.e., nifedipine/Cd-sensitive current traces, are shown in Fig. 1, B and C. In Ca2+-free solution (Fig. 1C), decay of the current was much slower than observed in the case of the Ca2+ current (Fig. 1B), and the tail inward current was observed. Inactivation process of the current was best fitted by functions with a two-exponential process (Fig. 1C).


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  		Fig. 1.   Currents through cardiac L-type Ca2+ channels in the absence of extracellular Ca2+. A: plot of peak inward currents elicited by a pulse to +10 mV against time after the start of whole cell current recording. The membrane potential was held at -40 mV, and 300-ms pulses were applied at 5-s intervals. Below the plot is given the composition of the external solution. The inward currents were blocked by 2 µM nifedipine (nif) and 500 µM CdCl2. B: nifedipine/Cd-sensitive current observed in 1.8 mM CaCl2 containing solution. C: nifedipine/Cd-sensitive current observed in Ca2+-free solution is shown (top). Semilogarithmic plots of the current are shown (bottom). During the 300-ms test pulse, the first points deviate from the straight line (time constant of 445 ms, R = 0.947) and the difference is shown by lower set points, which can be fitted by a second exponential function with a time constant of 21 ms (R = 0.994). Each current was obtained from the experiment shown in A.

Figure 2A shows a typical example of the subtracted current traces recorded in Ca2+-free solution, at selected potentials. Nifedipine/Cd-insensitive currents in each potential are presented in Fig. 2A. During depolarization, the inward currents began to activate at -20 mV (17 ± 9 pA), peaked at +10 mV or +20 mV (628 ± 38 pA for +10 mV, 605 ± 39 pA for +20 mV, n = 12), and reversed near +40 mV (see Fig. 6A). The reversal potential is similar to that observed by other researchers (18). The faster inactivating component was not remarkable up to -10 mV, and it appeared at 0 mV. At -10 mV, the decay of the current was best fitted by a function with a single-exponential process. Above 0 mV, the decay of the currents was best fitted by functions with a two-exponential process (Fig. 2B). Time constants of the faster inactivating component decreased with depolarization of membrane potentials (P < 0.008); 30.5 ± 2.2 ms at 0 mV, 23.1 ± 1.0 ms at +10 mV, 21.4 ± 1.0 ms at +20 mV, and 17.0 ± 1.5 ms at + 30 mV (n = 12; Fig. 3). There were no statistical significant differences between time constants at +10 mV and at +20 mV. Time constants of the slower inactivating component were not significantly affected by the membrane potential; 548 ± 15 ms at -10 mV, 489 ± 21 ms at 0 mV, 534 ± 27 ms at +10 mV, 598 ± 27 ms at +20 mV, and 530 ms ± 30 ms at +30 ms (n = 12; Fig. 3).


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  		Fig. 2.   Inactivation properties of currents through cardiac L-type Ca2+ channels in the absence of extracellular Ca2+. A: nifedipine/Cd-insensitive superimposed current traces recorded at membrane potentials of -30 mV-0 mV (upper left) and +10 to +30 mV (upper right). Nifedipine/Cd-sensitive superimposed current traces recorded at membrane potentials of -30 mV-0 mV (lower left) and +10 to +30 mV (lower right). These current traces were elicited by 300-ms test pulses at 5-s intervals from a holding potential of -40 mV in Ca2+-free solution. B: semilogarithmic plots of the currents shown in A. At -10 mV, the points were fitted by a straight line (time constant of 456 ms). Above +10 mV, in each panel, the first points deviate from the straight line, and the difference is shown by the lower set points, which can be fitted by a second exponential function.



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  		Fig. 3.   Mean values of the time constant of the faster inactivating components () and the slower inactivating components (open circle ) at presented membrane potentials in Ca2+-free solution. The left vertical axis is for the time constant of the slower inactivating component, and the right vertical axis is for the time constant of the faster inactivating component. Data points are means ± SE (n = 12).

Effects of isoproterenol on Na+ currents through the cardiac L-type Ca2+ channel. Application of isoproterenol (10 nM) increased peaks of the currents recorded at +10 mV, as shown in Fig. 4A. During the voltage-clamp step, the increase in peak current on isoproterenol was associated with a marked slowing of the time course of inactivation (Fig. 4B). As shown in Fig. 4B, isoproterenol slightly slowed the time course of activation. In the inactivation process, isoproterenol increased the slower inactivating component and decreased the faster inactivating component (Fig. 4C). We obtained similar results in two other experiments. To confirm these effects of isoproterenol on the inactivating process, we used 100 nM isoproterenol.


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  		Fig. 4.   Effects of isoproterenol (10 nM) on currents through cardiac L-type Ca2+ channel in Ca2+-free solution. A: plot of peak inward currents elicited by a pulse to +10 mV. The membrane potential was held at -40 mV, and 300-ms pulses were applied at 5-s intervals. Below the plot is given the composition of the external solution and the timing of application of isoproterenol. The inward currents were blocked by 2 µM nifedipine and 500 µM CdCl2. B: at left, superimposed current traces were obtained during the application of isoproterenol. At right, current in the absence of isoproterenol (normalized control) was scaled to a similar peak current amplitude recorded in the presence of isoproterenol. Nifedipine/Cd-insensitive currents were not subtracted in the case of these currents. C: at left, the isoproterenol increased-nifedipine/Cd-sensitive current. Semilogarithmic plots of the current are shown (right). During the 300-ms test pulse, the first points deviate from the straight line (time constant of 703 ms), and the difference is shown by the lower set points, which can be fitted by a second exponential function with a time constant of 25 ms. In B and C, each current trace was obtained from the experiment shown in A.

Figure 5A shows a typical example of the subtracted current traces recorded in the presence of 100 nM isoproterenol, at selected potentials. Nifedipine/Cd-insensitive currents in each potential are presented in Fig. 5A; isoproterenol increased the nifedipine/Cd-insensitive currents. During depolarizations, the inward current began to activate at -20 mV, peaked at 0 mV or +10 mV, and reversed near +40 mV (also see Fig. 6A). Figure 6A shows the peak current-voltage relationship of nifedipine/Cd-sensitive currents obtained in the absence and presence of isoproterenol. Enhancement of the currents was prominent, with weak depolarization. The relative values for the mean peak currents were 12.3, 4.2, 1.3, 1.4, 1.4, and 1.4 at -20 mV, -10 mV, 0 mV, +10 mV, +20 mV, and +30 mV, respectively (n = 12 for control, n = 8 for isoproterenol). Isoproterenol did not change the reversal potential of the currents. In the presence of 100 nM isoproterenol, the faster inactivating component was never observed at any membrane potential (Fig. 5, A and B). At -20 mV, during the 300-ms depolarization, the current did not inactivate (Fig. 5A). Although isoproterenol remarkably slowed the time course of activation in the cases of weak depolarizations, the effect of isoproterenol on activation at a higher membrane potential was not remarkable. Time course of the inactivating component observed in the presence of isoproterenol tended to slow with depolarization of membrane potentials; 591 ± 54 ms at -10 mV, 538 ± 60 ms at 0 mV, 642 ± 59 ms at +10 mV, 684 ± 38 ms at +20 mV, and 696 ± 65 ms at +30 mV (n = 8; Fig. 6B). There was no statistical significance between these values, and those at each test potential were not significantly different from those in the case of the slower inactivating components observed in the absence of isoproterenol.


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  		Fig. 5.   Effects of isoproterenol (100 nM) on inactivation properties of currents through cardiac L-type Ca2+ channel in Ca2+-free solution. A: nifedipine/Cd-insensitive superimposed current traces recorded at membrane potentials of -30 mV-0 mV (upper left) and +10 to +30 mV (upper right). Nifedipine/Cd-sensitive superimposed current traces recorded at membrane potentials of -30 mV-0 mV (lower left) and +10 to +30 mV (lower right), respectively. These current traces were elicited by 300-ms pulses at 5-s intervals from a holding potential of -40 mV in Ca2+-free solution with 100 nM isoproterenol. B: semilogarithmic plots of the currents shown in A. At each potential, the points during the whole of the 300-ms pulse can be fitted by a single exponential function.



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  		Fig. 6.   A: voltage dependence of peak nifedipine/Cd-sensitive currents in the absence (open circle ) and presence () of 100 nM isoproterenol. Depolarizations of 300-ms duration were present in 10-mV increments at 5-s intervals from a holding potential of -40 mV in Ca2+-free solution. Data points are means ± SE (n = 12 for control, n = 8 for isoproterenol). B: mean values of the time constant of the inactivating components in the presence of 100 nM isoproterenol at selected potentials. Data points are means ± SE (n = 8).

Effects of a protein kinase inhibitor and cAMP on Na+ currents through the cardiac L-type Ca2+ channel. Application of H-89 (10 µM) for 4 min decreased the currents recorded at +10 mV, as shown in Fig. 7A. The peak amplitude of the inward currents decreased to 52 ± 4% (n = 7) of the control value during application of H-89 for 4 min. The time course of inactivating components was not significantly affected by H-89. The time constants of the faster inactivating components were 27.6 ± 1.8 ms and 27.1 ± 2.7 ms for control and H-89, and the time constants of the slower inactivating components were 723 ± 68 ms and 652 ± 88 ms for control and H-89, respectively. The magnitudes of the faster inactivating component and the slower inactivating component showed different sensitivities to H-89. Initial amplitudes of the faster inactivating component and the slower inactivating component decreased to 72 ± 6% and 39 ± 7% of control values, respectively. In the presence of H-89, the effects of isoproterenol (100 nM) were observed. The peak amplitude of inward currents increased by 1.15 over that observed just before the application of isoproterenol in the presence of H-89 (n = 5). As shown in Fig. 7A, effects of isoproterenol in the presence of H-89 on the inactivating process differed from those observed in the absence of H-89. The inactivating process was fitted by functions with a two-exponential process. The time course of the slower inactivating component was accelerated with isoproterenol (346 ± 31 ms, n = 5, P < 0.01), but the time course of the faster inactivating component did not change significantly (30.5 ± 0.7 ms).


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  		Fig. 7.   Effects of a protein kinase inhibitor, H-89 (10 µM), and cAMP (500 µM), on inactivation properties of currents through cardiac L-type Ca2+ channel in Ca2+-free solution. Current traces were elicited by 300-ms pulses at 5-s intervals from a holding potential of -40 mV in Ca2+-free solution. A: at left, superimposed current traces were obtained during the sequential application of H-89 and isoproterenol (100 nM)/H-89 in a cell. Each current trace was obtained before H-89 (control), 4 min after 10 µM H-89, and at maximal effects of 100 nM isoproterenol in the presence of H-89. Isoproterenol was applied 4 min after the application of H-89. B: current trace obtained in 500 µM cAMP in the pipette solution is shown (left). Semilogarithmic plots of the current are shown (right). The points during the whole of the 300-ms pulse can be fitted by a single exponential function. C: current trace obtained in case of 5 µM isoproterenol is shown (left). Semilogarithmic plots of the current are shown (right). The points during the whole of the 300-ms pulse can be fitted by a single exponential function. In A-C, nifedipine/Cd-insensitive currents were subtracted. D: bar graph compares effects of 100 nM isoproterenol in the presence of 10 µM H-89 (n = 5), 100 nM isoproterenol (n = 8), and 500 µM cAMP (n = 6) on relative initial amplitude of the slower inactivating component. Relative initial amplitude of the slower inactivating component: initial amplitude of slow-inactivating component/(initial amplitude of fast-inactivating component + initial amplitude of slow-inactivating component). Data are means ± SE.

As shown in Fig. 7B, in the presence of 500 µM cAMP in the pipette solution, a large inward current with a single inactivating process was observed at +10 mV. The inactivating process was best fitted by a single-exponential function having a time constant of 1,343 ± 32 ms (n = 6). This value was significantly larger than that observed in the presence of 100 nM isoproterenol (Fig. 6B) and similar to that observed in the presence of 5 µM isoproterenol (Fig. 7C, 1,277 ± 116 ms, n = 3).

The effects of isoproterenol, cAMP, and H-89 on the inactivating process at +10 mV are summarized in Fig. 7D. The relative initial amplitudes of the slower inactivating component [initial amplitude of slow-inactivating component/(initial amplitude of fast-inactivating component + initial amplitude of slow-inactivating component)] were 0.37 ± 0.1 (n = 12), 0.43 ± 0.12 (n = 5), 1.0 (n = 8), and 1.0 (n = 6) for control, 100 nM isoproterenol with 10 µM H-89, 100 nM isoproterenol, and 500 µM cAMP, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results clearly show that the inactivation process of inward currents, carried by Na+ through the cardiac L-type Ca2+ channel in the absence of external Ca2+, consisted of two components. We first investigated the nature of the inactivating process of the two components. The inward currents began to activate at -20 mV, and the faster inactivating component appeared >0 mV. The time course of the faster inactivating component accelerated with depolarizing test potentials. On the other hand, time course of the slower inactivating component was not significantly affected by test potentials. Faster inactivating components have been observed in Ba2+ or Na+ currents through the cardiac L-type Ca2+ channel in whole cell recordings (3, 10); the voltage dependency and underlying mechanism were not given. We observed Na+ currents through the Ca2+ channel in the presence of 0.5 mM MgCl2, resulting in relatively small inward currents due to the blocking effect of Mg2+ (6, 9, 18). The Mg2+ block cannot explain the nature of the two inactivation processes, because the rates of Mg2+ block and unblock are very fast (16).

Isoproterenol increased the slower inactivating component and decreased the faster inactivating component, and a protein kinase inhibitor attenuated these effects. These results indicate that phosphorylation of the channel converts the faster inactivating component into the slower one. In single-channel studies, phosphorylation of the channel via the beta -receptor-cAMP cascade modulates slow-gating kinetics and prolongs duration of the available state, in which the channel can open with membrane depolarization (4, 21, 27). Yue et al. (27) found that the stimulation of the beta -receptor-cAMP cascade also remarkably prolonged open time of the channel, indicating that phosphorylation modulates rapid-gating kinetics. These effects on the channel result in an increase in the current and slowdown in inactivation of the macroscopic Ca2+ channel current (19). The faster inactivating component and the slower inactivating component showed different sensitivities to a protein kinase inhibitor in that the slower inactivating component was decreased more remarkably by H-89 than was the faster inactivating component. Similar findings were noted in the case of Ca2+ currents in guinea pig ventricular myocytes where a chemical phosphatase decreased the slower inactivating component rather than the faster inactivating one (1). Reduction in the faster inactivating component suggests that the component represents kinetics of a partly phosphorylated channel. Although it has been reported that the Ca2+ channel cannot open without phosphorylation (14, 24), some investigators have reported that phosphorylation is necessary for channel opening activity (8, 22). In the presence of intracellular cAMP or a relatively high concentration of isoproterenol, inactivation of the currents consisted of a single slow process with a very slow time course. Ono and Fozzard (22) have reported that a high concentration (16 µM) of isoproterenol induced channel openings with long open time.

The above arguments lead to the hypothesis that these three components (the faster inactivating component, the slower inactivating component, and the very slow inactivating component) correspond to the degree of phosphorylation of the channel: the minimally phosphorylated channel shows faster inactivation, and the intermediately phosphorylated channel shows slower inactivation. Finally, the maximally phosphorylated channel shows very slow inactivation. This hypothesis is consistent with proposals concerning single-channel studies (8, 22).

Application of isoproterenol increased the peak currents to a greater extent with weak depolarizations, findings compatible with data in the case of Ba2+ currents in rat cardiac myocytes (26) and in frog ventricular cells (2). The leftward shift of activation properties probably contributes to this voltage-dependent enhancement (2, 26). According to our present data, difference in the activation threshold between the faster inactivating component and the slower inactivating one may be responsible for the voltage-dependent effect of isoproterenol: isoproterenol may convert the faster inactivating component, which has a high threshold of activation, into the slower inactivating component, which has a low threshold of activation, the result being a large enhancement in weak depolarization. These arguments are compatible with the shift in cardiac Ca2+ channel gating currents by isoproterenol, as noted in embryonic chick heart cells (11). In the case of inactivating properties, the effects of isoproterenol cannot be explained by the shift, because time constants of the faster inactivating components (17 ms ~ 30.5 ms) are not comparable to those of the inactivating components in the presence of 100 nM isoproterenol (538 ms ~ 696 ms). We found that isoproterenol slowed the time course of activation in the case of weak depolarizing test potentials, an event noted in frog ventricular cells and in cultured neonatal rat ventricular cells (2).

We fitted the inactivation time course of the faster component after fitting that of the slower component. To some extent, this procedure affects the time constant of the faster inactivating component. For a more accurate time constant of inactivating component, it will be necessary to use longer test pulses for currents reaching a steady-state level.

Our data are in agreement with previous observations of single-channel studies and whole cell studies, and present a new insight into inactivating kinetics of macroscopic currents through phosphorylated and dephosphorylated channels. On the basis of our results, it will be possible to identify phosphorylation sites responsible for changing the inactivation process, using heterologously expressed cardiac L-type Ca2+ channel subunits with site-directed mutagenesis.


    ACKNOWLEDGEMENTS

We thank Drs. M. Kameyama and K. Yamaoka for helpful discussion.


    FOOTNOTES

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.

Address for reprint requests and other correspondence: M. Kaibara, Dept. of Pharmacology, Nagasaki Univ., School of Medicine, 1-12-4 Sakamoto, Nagasaki 8528523, Japan (E-mail: mkaibara{at}alpha.med.nagasaki-u.ac.jp).

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 3 May 1999; accepted in final form 16 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 279(3):C603-C610
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