Centre National de la Recherche Scientifique Unité Mixte de Recherche 6542, Faculté des Sciences, Université de Tours, 37041 Tours Cedex, France
Submitted 13 August 2002 ; accepted in final form 26 March 2003
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ABSTRACT |
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L-type calcium current; calcium-dependent inactivation; facilitation; phosphorylation; cesium
Ca2+ current can be upregulated through different
mechanisms. The best known of these is phosphorylation of the channel by a
cAMP-dependent protein kinase (PKA) following stimulation of -adrenergic
receptors (9). During an action
potential, increased entry of Ca2+ following the
phosphorylation of DHPrs has to be regulated to avoid
Ca2+ overload. This is done through different mechanisms
such as increased K+ current and
Ca2+-dependent inactivation of the DHPr
(5). Another mechanism that
leads to Ca2+ current upregulation is facilitation. In
cardiac cells an increase in the frequency of activation of
Ca2+ channels over a physiological range (0.55
Hz) induces an increase of the Ca2+ current
(11,
21,
29). Recently it was also
shown (3) that moderate
depolarization of the diastolic membrane potential also promotes facilitation
of ICaL, which is enhanced by intracellular cAMP.
The aim of the present study was to investigate the mechanism that underlies multiphasic inactivation of ICaL that was recorded in our experiments. In vitro patch-clamp studies of calcium current usually seek to avoid contamination by other currents by substituting physiological Na+ and K+ by impermeant ions like Cs+. We show that intracellular Cs+ activates the PKA-dependent pathway leading to the phosphorylation of the DHPr and/or the RyR and/or the regulatory protein of the SR Ca2+-ATPase (SERCA), phospholamban (PLB). Such phosphorylation underlies very fast and reversible Ca2+-dependent inactivation of ICaL.
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MATERIALS AND METHODS |
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Solutions. Experimental solutions are shown in Table 1. Extracellular test solutions were applied by using a multiple microcapillary perfusion system (19) placed in close proximity to the cell (0.5 mm). All salts and drugs were purchased from Sigma Chemicals, except H-89, which was obtained from Calbiochem. CdCl2, 4-aminopyridine (4-AP), and caffeine were added to the external solutions at the concentrations given. Stock solutions of tetrodotoxin (TTX; 1 mmol/l), cyclopiazonic acid (CPA; 10 mmol/l), ryanodine (10 mmol/l), H-89 (1 mmol/l), forskolin (1 mmol/l), and isoproterenol (1 mmol/l) were prepared in distilled water and added to the external solutions to provide the concentrations indicated. Experiments involving the use of nifedipine (100 µmol/l, stock solution 10 mmol/l in ethanol) were performed in the dark. Protein kinase inhibitor (PKI; 200 nmol/l, stock solution 1 mmol/l in distilled water) was included in the internal solution of one set of experiments (see Fig. 5).
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Cell current measurements. Ca2+ currents
were recorded using the whole cell configuration of the patch-clamp technique.
In most of the experiments, conditions were optimized to eliminate currents
other than ICaL by using Na+-K+-free
solutions (see Table 1,
Na+-K+-free extracellular and Cs+
intracellular solutions). In some experiments (e.g., see Figs.
6 and
7), different solutions were
used to record ICaL
(Table 1). Junction potentials
between the intrapipette solution and the reference electrode were canceled
before tight seals were obtained. The pipettes were obtained from borosilicate
glass tubes (GC150F-15; Clark Electromedical Instrument) with a puller (PC103;
Narashige, Tokyo, Japan) and slightly fire-polished (MF-9; Narashige). When
filled with the pipette solution, pipette resistance was 1.53 M.
In some experiments, pipettes were coated with dental wax and pipette and cell
capacitance were compensated. The series resistance was compensated by
5070%. In other experiments, voltage errors resulting from
uncompensated series resistance were <5 mV and were not corrected. Cell
membrane capacitance was measured by integrating the capacitance current
recorded during a 10-mV hyperpolarizing pulse from a holding potential of -80
mV.
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The voltage clamp circuit was provided by an RK 400 patch-clamp amplifier (Biologic, Grenoble, France). Voltage parameters were controlled by a Pentium PC connected through a DIGIDATA 1200 analog interface (Axon Instruments, Burlingame, CA). Data acquisition and analysis were accomplished using pCLAMP software (Axon Instruments). Sampling frequency was 10 kHz, and signals were filtered at 3 kHz with the use of an 8-pole Butterworth filter before acquisition.
ICaL was elicited with 300- or 500-ms voltage steps to 0 mV from a holding potential of -80 mV at a frequency of 0.125 Hz. ICaL was measured as the difference between the peak inward current and the current at the end of the depolarizing pulse. Currents are expressed in current density (pA/pF). Current-voltage (I-V) relationships and availability (steady-state inactivation) curves were obtained with a double pulse protocol that consisted of 500-ms-duration prepulses from -80 to +60 mV in 10-mV steps, a 5-ms interpulse interval at -80 mV, and a test pulse to 0 mV for 500 ms. The stimulus frequency was 0.125 Hz. Availability curves were obtained by normalization of the amplitude of the test pulse current to its amplitude recorded in the absence of a prepulse.
Results are expressed as means ± SE (n = no. of cells). Statistical differences were assayed by using one- or two-way ANOVA as appropriate, followed by the post hoc Dunnett's test. Statistics were performed by using SigmaStat version 1.0 software.
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RESULTS |
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We examined various alternative explanations for this variability of the kinetics of ICaL from different myocytes. This variability was observed in different cells obtained from the same batches of cell isolations, so it was not a consequence of the quality of the cell isolation procedure. Interestingly, complex inactivation of ICaL was always prominent at negative voltages (-20 mV in Fig. 1B), whereas it appeared as a break point or notch between two distinct phases of decay of ICaL between -10 and 10 mV. We rejected any methodological problems such as voltage clamp control or lack of clamp uniformity provoking this irregularity because, first, the I-V curves had the same shape whether decay was simple or complex (Fig. 1C), and second, if there was loss of voltage control, the irregularity would be more prominent at positive voltages where the current was larger. It was verified that series resistance and cell capacitance compensation up to 70% did not affect the shape of the current (n = 10).
Despite the lack of a shoulder in the I-V relationships at negative potentials, we looked for possible contamination of ICaL by the fast Na+ current (INa; notwithstanding that the external solution was Na+ free), the TTX-sensitive Ca2+ current (14), and the T-type Ca2+ current (ICaT). The addition of 30 µM TTX to the external solution had no effect on the shape of recorded ICaL (n = 6). The addition of 40 µM Ni2+, a concentration known to block ICaT without affecting ICaL (26), had no effect (n = 6; Fig. 2A). Transient outward K+ currents (ITO), which are present in cardiac cells from other species, are sensitive to 4-AP and/or to intracellular Ca2+. Although our experiments were conducted with a Cs+-rich intracellular solution that did not contain K+, we tested the effect of the addition of 2 mM 4-AP, which blocks Ca2+-insensitive transient outward currents. This had no effect (n = 9; Fig. 2B). Also a transient outward current was not revealed when either external Ca2+ was removed from the medium (n = 2) or when ICaL was blocked by an inorganic blocker (200 µM Cd2+, n = 5; data not shown) or by an organic blocker (100 µM nifedipine, n = 8; Fig. 2C). We next examined whether calcium ions had to flow through the Ca2+ channels to induce multiphasic inactivation. Figure 2D shows that, after substitution of Ca2+ by Ba2+, notched decay of the current was no longer visible (n = 6). Also, complex inactivation of ICaL was not observed in experiments where BAPTA replaced EGTA in the pipette solution (n = 10; Fig. 2E).
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These last results suggest that multiphasic decay of the current resulted from Ca2+ influx and accumulation beneath the membrane.
Origin of the Ca2+ dependence of complex decay of ICaL. In the following experiments, the involvement of the SR in multiphasic inactivation of the L-type Ca2+ current in Cs+-loaded myocytes due to Ca2+-induced Ca2+ release (CICR) was tested. In the presence of 100 µM ryanodine, the Ca2+ release channel (RyR) of the SR is unable to release Ca2+ stored in the SR, whereas sarcolemmal L-type Ca2+ channels are unaffected (18, 23). Figure 3A shows that the application of 100 µM of ryanodine induced the disappearance of notched decay, a slowing down of the inactivation phase of the current, and an increase of the current amplitude. In addition, prepulse voltage facilitation disappeared with ryanodine (Fig. 3B). Similar results were obtained after the application of either 100 µM CPA (n = 3), which prevents Ca2+ loading of the SR (37), or 10 mM caffeine (n = 5), which causes emptying of the SR. The notched decay of ICaL was therefore a direct consequence of Ca2+ released from the SR.
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The notched and multiphasic decay of ICaL were more
prominent at negative voltages (Fig.
1A). To further characterize this relationship, we
evaluated the CICR-sensitive part of the current by subtracting control
currents from those recorded after the application of 100 µM ryanodine
(Fig. 4A).
Interestingly, the form of these difference currents exhibited kinetics
similar to estimated SR Ca2+ release fluxes from groups
of RYR (36). The mean
(time constant) for relaxation of the SR-sensitive current was 14 ± 1
ms (single exponential fitting, n = 6). The amplitude of the
SR-sensitive current decreased with depolarization
(Fig. 4B).
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The dependence of multiphasic decay of ICaL on intracellular Ca2+ and CICR could arise in one of two ways. First, the increase in intracellular Ca2+ could activate a Ca2+-dependent Cl- current [ICl(Ca)], which would distort the recording of ICaL and provoke an apparently complex decay resulting from the superposition of two different currents. Second, the increase in intracellular Ca2+ could provoke the decay of ICaL via Ca2+-induced inactivation, which would therefore represent a rapid and transient process that could be reversible on a time scale of tens of milliseconds.
ACa2+-activated Cl- current has been characterized in rabbit, dog, and mouse cardiac cells (4144). In our experimental conditions with the Na+-K+-free extracellular solution and the Cs+-based intracellular solution described in Table 1, the equilibrium potential for chloride (ECl) would be about -2 mV. In these circumstances a Cl- current would appear as an inward current at negative membrane potentials and as an outward current at positive membrane potentials. The ryanodine-difference currents shown in Fig. 4 did not reverse direction at or near to 0 mV. They were outwardly directed at all voltages with no signs of reversing; instead, they approached zero asymptomatically with depolarization. If the influx of Ca2+ via ICaL and CICR were to have evoked ICl(Ca), this would have appeared during voltage steps to negative membrane potentials as a reduction of the decay of ICaL, which would then be accelerated when ryanodine blocked CICR. In the recordings shown in Fig. 3A, the opposite was true. Sipido et al. (34) also rejected contamination of the recording of ICaL by ICl(Ca) in guinea pig ventricular myocytes. We conclude that multiphasic or notched decay of ICaL resulted from Ca2+-induced inactivation of the current that was provoked by CICR.
Modulation of ICaL inactivation by a phosphorylation pathway. The next question that we addressed was why, irrespective of identical basal experimental conditions with Na+-K+-free external and Cs+-rich internal solutions, some myocytes showed an enlarged ICaL amplitude, multiphasic ICaL decay, and prepulse diastolic facilitation, whereas others showed apparently normal control ICaL? Ca2+ cycling in cardiac cells is known to be very sensitive to phosphorylation, and Barrere-Lemaire et al. (3) showed that the diastolic depolarization-induced facilitation is enhanced by cAMP-dependent phosphorylation. We therefore postulated that the PKA pathway might be active in basal conditions in some cells in the absence of external agonist stimulation. To test this, we superfused cells that showed notched decay of ICaL with a solution that contained 5 µM of the PKA inhibitor H-89. Figure 5A shows that this reduced current amplitude, slowed decay (Fig. 5A, inset), and abolished prepulse voltage-dependent facilitation (Fig. 5B, n = 7). To confirm these results, we used a more specific PKA inhibitor, the PKI peptide. This peptide was included in the pipette solution, and a representative result is presented in Fig. 5C. The amplitude of ICaL gradually decreased with time, and this was associated with the disappearance of the notch (n = 3). Interestingly, the effect on the inactivation phase was more marked with PKI than H-89. This result could be explained by the fact that phosphorylation of PLB may be less sensitive to the effect of H-89 (15). To check that this decrease of ICaL over time is not due to rundown, we performed the same experiments without PKI in the pipette solution, and no significant decrease of ICaL was observed (Fig. 5C, n = 7).
We conclude from these experiments that the PKA-dependent phosphorylation pathway was active in basal conditions, leading to phosphorylation of the L-type Ca2+ channel and/or of the RyR and/or PLB.
Influence of the intracellular medium on inactivation of
ICaL. The next series of experiments was designed to
elucidate the mechanism responsible for the apparently high level of
phosphorylation in our basal experimental conditions. Vargas et al.
(39) showed that
Cs+ increased PKA activity in vitro. We first compared results from
cells in which N-methyl-D-glucamine (NMDG) rather than
Cs+ replaced intracellular K+
(Table 1). In basal conditions
ICaL never showed notched or multiphasic decay when the
myocytes were loaded with NMDG-rich intracellular solution
(Fig. 6B).
Table 2 shows that under basal
conditions, ICaL in Cs+-loaded cells with
notched decay was significantly larger than in those without notched decay,
whether they were loaded with Cs+ or NMDG (P < 0.05).
The responses of cells with these different intracellular media to
-adrenergic stimulation were tested.
Figure 6A shows a
Ca2+ current elicited at -10 mV in a cell with
Cs+-based pipette solution. This cell clearly showed a notched
decay under basal conditions, and the addition of 1 µM isoproterenol had
only a weak effect on ICaL amplitude. In 11 similar cells,
the amplitude of the current was increased significantly by 55 ± 18% by
addition of 1 µM isoproterenol. Figure
6B shows a Ca2+ current evoked in a
cell with an NMDG-rich pipette solution. In basal conditions the current did
not show notched decay, and the amplitude of the current was strongly
increased by addition of 1 µM isoproterenol (168 ± 23%, n =
6, P < 0.05 compared with notched ICaL). The
response of ICaL to isoproterenol in cells with either
Cs+- or NMDG-rich intracellular solutions is summarized in
Fig. 6C. Cells with
multiphasic inactivation of ICaL, although larger under
control conditions than in their counterparts with simple decay of
ICaL, responded poorly to isoproterenol. Interestingly,
cells with monophasic inactivation of ICaL responded to
isoproterenol stimulation with an increase in ICaL
amplitude of 207 ± 36% (n = 14). As found with NMDG-rich
pipette solution, after isoproterenol stimulation ICaL
inactivation showed a multiphasic inactivation. To confirm the role of the PKA
pathway, we used forskolin to directly stimulate the adenylyl cyclase. We
found that the amplitude of ICaL showing multiphasic
inactivation increased by only 58 ± 8% (n = 6) with forskolin,
whereas in cells presenting ICaL with monophasic decay,
ICaL increased by 178 ± 18% (n = 15; data
not shown). Forskolin also induced the appearance of the notch during the
inactivation phase (n = 14 of 15 cells).
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To further investigate the incidence of complex decay of ICaL consecutive to activation of the PKA pathway, we investigated the effects of isoproterenol on ICaL in more physiological conditions with a Na+-rich extracellular solution and a K+-rich intracellular solution (Table 1). Figure 7A shows Ca2+ currents elicited by voltage steps to -40, -20, and 0 mV in a representative cell. In control conditions the decay of the current was always simple (Fig. 7Aa). The application of isoproterenol induced a large increase of Ca2+ current amplitude (Fig. 7B) and notched or multiphasic decay (Fig. 7Ab). As found in the previous experiments with Na+-K+ free extracellular and Cs+-rich intracellular solutions (Fig. 3), multiphasic decay of ICaL in cells with a K+-rich intracellular solution was due to Ca2+ released from the SR, because the addition of ryanodine to the external solution slowed inactivation, removed multiphasic decay (Fig. 7Ac), and further increased current amplitude (Fig. 7B). In basal conditions ryanodine had no effect on current amplitude (Fig. 7B).
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DISCUSSION |
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Intracellular Cs+ and excitation-contraction studies. Most of the observations here correspond to a particular, but widely used, experimental condition where ICaL is isolated by replacing intracellular K+ by Cs+ (1, 2, 26). Recently, however, Cs+ has been shown to significantly increase the activity of the catalytic subunit of PKA (39). Though our experiments did not directly measure PKA activity, we recorded the activity of one of the effectors of this pathway, the L-type Ca2+ current. In the majority of cells infused with a Cs+-rich pipette solution under otherwise basal conditions, the application of PKA inhibitors (H-89, PKI) reduced current amplitude and slowed inactivation, causing the disappearance of notched or multiphasic decay. Also, in these cells isoproterenol had only a small effect on the amplitude of ICaL. It was as if the current had already been activated by this signaling pathway, which concords with its greater amplitude under basal conditions. Evidence that these results are due to Cs+ and not to the replacement of intracellular K+ was shown when cells were infused with NMDG-rich pipette solution, where ICaL was smaller, with normal current decay under basal conditions and greater responsiveness to isoproterenol. Similar results were obtained when quasi-physiological solutions were used in the recording of ICaL. Together, these results strongly suggest that intracellular Cs+ activates the PKA pathway.
Furthermore, it has been proposed that intracellular Cs+ blocks K+ channels present on the SR membrane and thereby alters Ca2+-cycling properties of cardiac cells (17, 22). Our finding and these findings indicate that care must be taken when Cs+ is used instead of K+ in the intracellular solution to study excitation-contraction (EC) coupling in cardiac myocytes.
Fast and reversible Ca2+-dependent inactivation of ICaL. Another result of this study was that in conditions when the PKA pathway was activated, the local release of Ca2+ by the SR (32) was able to rapidly and reversibly inactivate ICaL, which was responsible for the appearance of either multiphasic or notched decay of ICaL. The increase in the initial phase of decay was probably due to an increased Ca2+ concentration underneath the membrane after enhanced SR Ca2+ release. The recovery of ICaL during the maintained voltage step that gave rise to multiphasic decay was probably due to the reduction of Ca2+ concentration in the subsarcolemmal microdomain by diffusion and reuptake by the SR. The channel then came back from the Ca2+-inactivated state to the open state. The Ca2+ uptake occurs via the SERCA, which must belong to the microdomain. Therefore, DHPr, RyR, and SERCA belong to the same Ca2+ microdomain, not influenced by the presence of the Ca2+ chelator EGTA. Calcium microdomains are known to exist in the rat ventricular cell, but our observations are interesting because the guinea pig myocyte bears more similarity to the human cardiac myocyte (5). With BAPTA, the notch was never observed, suggesting that the accessibility of EGTA is not sterical but, more likely, limited to the speed necessary to chelate Ca2+ (25). Further studies are required to determine which of the key proteins was phosphorylated by Cs+ activation of PKA: the DHPr, the RyR, or the regulatory protein PLB (6).
If reversible inactivation were directly linked to the local control of
Ca2+ release, this would assume a local release of
Ca2+ from the SR that was tightly synchronized to the
peak ICaL with a duration of the local SR
Ca2+ release event between 7 and 30 ms
(5,
36). The fact that notched or
multiphasic decay was more prominent at negative membrane potentials agrees
with the gain of CICR (40),
which increases with the level of phosphorylation
(12). Thus these experiments
provide new evidence that fast negative feedback from the release of
Ca2+ from the SR to ICaL via
Ca2+-dependent inactivation occurs proportionally to the
amplitude of the triggering signal. In this manner, the increase of
ICaL upon activation of the PKA phosphorylation pathway
would permit the fast and reversible control of EC coupling. This mechanism
would play an important role during -adrenergic stimulation of the
heart. Finally, it is important to note that those cells that lacked complex
inactivation of ICaL under basal conditions acquired it
upon the application of isoproterenol. This corroborates the hypothesis that
fast inactivation of ICaL mediated by Ca2+
release from the SR requires PKA-dependent phosphorylation.
Low diastolic depolarization facilitation. Diastolic potential-induced facilitation of the ICaL was only observed under basal conditions in this study when Cs+ was used to substitute for K+. Barrere-Lemaire et al. (3), who also used Cs+ in their pipette solution, suggested that this phenomenon was linked to CICR Ca2+-induced inactivation of the L-type Ca2+ current favored by cAMP-dependent phosphorylation. Thus during the prepulse, either a release of Ca2+ from the SR occurred, leading to reduced Ca2+-dependent inactivation during the subsequent test pulse, or the channel changed to a Ca2+-insensitive conformation. Whatever the mechanism, it required the PKA phosphorylation of the L-type Ca2+ channel and/or the RyR and/or PLB. It is interesting to note that similar properties have been described for frequency-dependent facilitation, e.g., increase in the amplitude and slowing of the inactivation phase of ICaL (11).
Some questions remain to be elucidated. Why would only certain cardiomyocytes, under basal conditions, show fast inactivation upregulated by the PKA phosphorylation pathway? Such a question probably finds an answer in the regulatory mechanism proposed by duBell et al. (12), who suggested that EC coupling, including ICaL, was a dynamic balance between phosphorylation and dephosphorylation. The two kinds of cells described here could therefore correspond to a difference in the equilibrium of such a dynamic process, exacerbated by the presence of Cs+ in the internal medium. Populations of cells with monophasic inactivation of ICaL could have a higher basal level of phosphatase activity, as indicated by the lower effect of isoproterenol and forskolin stimulation.
Why have other studies not reported this notched inactivation of ICaL in the same species? Some investigators used NMDG-rich intracellular solution (e.g., see Ref. 35). In the great majority of the studies in which Cs+ was used in the pipette solution, it is likely that investigators missed this point by using a depolarizing prepulse or holding potential (-40 mV) to inactivate INa and ICaT (e.g., see Refs. 4, 27, and 31). It is known that a nonphysiological depolarized holding potential interferes with the functioning of the L-type Ca2+ channels, inducing a slowing of the inactivation phase of ICaL (3, 38). Finally, many investigators have used depolarized test pulses of 0 or +10 mV at which the notch is less pronounced (Fig. 1B).
In conclusion, we have shown that intracellular Cs+ is able to activate the PKA pathway, modifying in consequence the activity of the targets of this kinase. We have shown that PKA activation enhances Ca2+-dependent inactivation of ICaL, which then closely follows the kinetics of localized variation in [Ca2+]. We have shown that diastolic potential-induced facilitation of ICaL is a phenomenon dependent on Ca2+-dependent inactivation, which also requires activation of the PKA pathway.
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ACKNOWLEDGMENTS |
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Present addresses: A. Lacampagne, Physiopathologie Cardiovasculaire, INSERM U390, CHU Arnaud de Villeneuve, 34295 Montpellier, France; L. Sallé, Laboratoire de Physiologie Cellulaire, Université de Caen, 14032 Caen Cedex, France; J.-Y. Le Guennec, Nutrition Croissance et Cancer, INSERM Emi 0211, Faculté de Médecine, 37032 Tours, France.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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