Effects of Protein Phosphatase and Kinase Inhibitors on Ca2+ and Cl− Currents in Guinea Pig Ventricular Myocytes

  1. Yoshiyuki Hirayama and
  2. H. Criss Hartzell
  1. Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030
     
    Next Section

    Abstract

    It is well-established that in heart, both the L-type Ca2+channel and the cystic fibrosis transmembrane conductance regulator Cl− channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (ICa and ICl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on ICl are about twice as fast as the effects on ICa, probably because the dephosphorylation reaction for ICl is faster than that for ICa. In contrast, inhibition of protein phosphatases with 10 μm microcystin stimulated both ICa and ICl, but the stimulation of ICl was much slower and smaller than the stimulation of ICa. The effect of microcystin was inhibited by staurosporine (Ki = 171.5 and 161 nm for ICa and ICl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC(19–31), and it was not PKA because it was not inhibited by adenosine 3′,5′-cyclic phosphorothioate. These results suggest that although both the Ca2+ and Cl− channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.

    In mammalian cardiac myocytes, β-adrenergic agonists regulate a variety of ionic currents, including ICa and ICl (1-3). Both currents are stimulated by cAMP-dependent phosphorylation via the Gs/adenylyl cyclase/cAMP/PKA cascade, but both of these currents may be regulated by phosphorylation of more than a single phosphorylation site.

    In the case of ICa, Tsien et al. (4) proposed that two different phosphorylation sites were responsible for the β-adrenergic regulation of Ca2+ channel availability (N) and channel gating (Po). Support for this hypothesis has come from studies on rabbit ventricular myocytes in which different concentrations of okadaic acid selectively affect N and po (5) and from our studies on frog ventricular myocytes in which the amplitude of ICa is regulated by two phosphorylation sites that can be distinguished by the phosphatases that dephosphorylate them (6). One site is dephosphorylated by phosphatase 2A, and the other site is dephosphorylated by a phosphatase with a low sensitivity to the phosphatase inhibitors microcystin, okadaic acid, and calyculin A.

    The regulation of ICl is even more complicated (3). The regulation of this channel, like that of the Ca2+ channel, involves two phosphorylation sites. One site is dephosphorylated by protein phosphatase 2A, and the other is dephosphorylated by a phosphatase with a low sensitivity to microcystin and okadaic acid, which may be protein phosphatase 2C (7). In addition, the channel is regulated by ATP binding and hydrolysis by its two nucleotide binding domains. The singly phosphorylated channel exhibits brief openings that correspond to hydrolysis of ATP by the first nucleotide binding site. The doubly phosphorylated channel exhibits a much longer channel open time that involves ATP binding to the second nucleotide binding site and stabilization of channel open state.

    Although it is clear that both channels are regulated by PKA, there is evidence that other protein kinases may also be involved in their regulation as well. For example, PKC can regulate cardiac Ca2+ channels (8), and the CFTR Cl− channel may also be regulated by PKC (3). Furthermore, we have recently shown that in frog ventricular myocytes there is a novel protein kinase, which we have termed PKX, that can phosphorylate one of the phosphorylation sites that regulates ICa (6).

    The fact that both ICa and ICl seem to be regulated by two phosphorylation sites and that multiple protein kinases can regulate these channels raises the question of whether these channels are regulated in concert or independently by phosphorylation. For example, when cAMP levels increase in the cell, do both channels become phosphorylated with the same kinetics, or can the phosphorylation of each channel be adjusted independently? One way that phosphorylation could be adjusted would be by alterations in the activity of the protein phosphatases responsible for dephosphorylation. For these reasons, we decided to compare the regulation of ICa and ICl by β-adrenergic agonists and inhibitors of protein phosphatases and kinases in guinea pig ventricular myocytes.

    Previous SectionNext Section

    Materials and Methods

    Isolation of cardiac myocytes.

    Single guinea pig ventricular myocytes were isolated by enzymatic dissociation. Female guinea pigs, weighing 300–400 g, were anesthetized with sodium pentobarbital (50 mg/kg). The chest was opened under artificial respiration, and the aorta was cannulated for perfusion with Tyrode’s solution before the heart was removed from the animal. After the perfusate was changed to nominally Ca2+-free Tyrode’s solution for ∼5 min, a nominally Ca2+-free Tyrode’s solution with 0.1–0.11 mg/ml collagenase (Yakult Co., Tokyo, Japan) was recirculated for ∼9 min using a Langendorff apparatus. After the enzyme was washed out, the hearts were then stored in high-K+ and low-Cl−solution at room temperature (22°) for 10 min. The isolated cells were obtained by gentle agitation of small pieces of ventricle and stored in the high-K+ and low-Cl− solution for 1 hr. They were then kept in Tyrode’s solution at room temperature before use.

    Solutions.

    The composition of the Tyrode’s solution was 144 mm NaCl, 0.33 mmNaH2PO4, 4.0 mmKCl, 1.8 mm CaCl2, 0.53 mm MgCl2, 5.5 mm glucose, and 5.0 mm HEPES-NaOH buffer, pH adjusted to 7.4 with NaOH. Nominally Ca2+-free Tyrode’s solution simply omitted CaCl2. High-K+ and low-Cl− solution contained 70 mmglutamic acid, 15 mm taurine, 30 mm KCl, 10 mm KH2PO4, 0.5 mm MgCl2, 11 mm glucose, 0.5 mm EGTA, and 5.0 mm HEPES-NaOH buffer, pH adjusted to 7.4 with KOH. The internal solution for recording ICa contained 85 mm CsCl, 10 mm EGTA, 20 mm tetraethylammonium Cl, 10 mm MgATP, 2 mm MgCl2, 5 mm pyruvic acid, 5 mmTris2-creatine phosphate, 0.1 mmTris-GTP, 5.5 mm glucose, and 10 mm PIPES, pH to 7.15 with CsOH. External solution contained 130 mm NaCl, 20 mm CsCl, 1.8 mm CaCl2, 0.5 mm MgCl2, 5.5 mmglucose, and 5 mm HEPES, pH to 7.3 with CsOH. In some experiments, 0.02 mm ouabain was added to suppress Na-K pump currents (9). In Fig. 5, the composition of the 158 mmCl− external solution was the same as the standard external solution except that 2 mmCoCl2 replaced the 1.8 mmCaCl2. The 28.6 mmCl− solution was the same as the 158 mm Cl− solution except that NaCl was replaced with Na-aspartate. The bath was continuously superfused with control solution at a rate of ∼2 ml/min, and the patch-clamped cell could be exposed to different test solutions within 1 sec by placing the cell at the mouth of one of a group of capillary tubes attached to reservoirs flowing at ∼100 μl/min.

    Figure 5
    View larger version:
    Figure 5

    Current-voltage relationship of the microcystin-stimulated background current. A, Current-voltage relationships obtained by triangular ramp pulses (−50 to +50 mV, dV/dt = 0.34 V/sec) before (a) and after (b) application of 10 μm microcystin. B, Current-voltage relationship of the microcystin-stimulated background current obtained after subtractinga from b in A. C, Effect of changing external [Cl−]o on current-voltage relations for the microcystin-stimulated background current. 1, 158 mm [Cl−]o. 2, 28.6 mm [Cl−]o. [Cl−]i was 109 mm. Erev at 158 mm[Cl−]o was −7.1 ± 1.9 mV. Erev at 28.6 mm[Cl−]o was +36.2 ± 3.9 mV. Values are mean of four or five cells. Dotted lines, current-voltage relation calculated from the Goldmann-Hodgkin-Katz equation assuming a pure Cl− conductance.

    The internal solution was changed as previously described (6). Briefly, a fine polyethylene tube was inserted to within 250 μm of the tip of the patch pipette. At the onset of the experiment, the other end of the tubing was placed in a reservoir containing control internal solution while the gigaohm seal was being made and broken. Measurements of control currents were made while control solution was being perfused into the pipette. Then, the negative pressure was released, and the tubing was switched to another reservoir containing the experimental drug dissolved in the control solution. The new solution was then aspirated into the patch pipette by again applying negative pressure to the patch pipette holder.

    Drugs.

    Reagents included cAMP-dependent protein kinase inhibitor peptide Rp-cAMPS (gift of Dr. Ira Cohen, State University of New York, Stony Brook, NY); microcystin-LR (GIBCO BRL, Gaithersburg, MD); K252a, staurosporine, and Fsk (Calbiochem, San Diego, CA); calyculin A (LC Laboratories, Woburn, MA); TPA (Sigma Chemical, St. Louis, MO); and myr-PKC(19–31)(Promega, Madison, WI). Stock solutions of staurosporine were prepared at 1 mm in dimethylsulfoxide. Microcystin was made at a concentration of 0.5 mm in 10% methanol. Fsk was 10 mm in 100% ethanol. Stock solutions were stored at −20°.

    Recording methods.

    Whole-cell currents were recorded using a patch-clamp amplifier (EPC-7; List Medical Instruments, Darmstadt, Germany). Cells were patch-clamped with borosilicate patch pipettes with a resistance of 1–2 MΩ. Total series resistance was usually <3 MΩ. ICa was elicited by voltage pulses delivered by a programmable digital stimulator (Challenger DB; W. Goolsby, Kinetic software and Emory University, Atlanta, GA). Routine pulses were from −50 to 0 mV. The pulse was 200 msec in duration, and ICa was measured as the peak inward current minus the current at the end of the pulse. Because the reversal potential of ICl (ECl) was measured to be −7 mV and calculated to be −10 mV and because ICl at −50 mV was small relative to ICa, contamination of ICaby ICl at 0 mV should be negligible. ICl was measured as the steady state current at −50 mV. Experiments were conducted at room temperature unless otherwise stated.

    Previous SectionNext Section

    Results

    Kinetics of stimulation of ICa and ICl by Iso.

    Initially, we were interested in comparing the time course of the effects of β-adrenergic stimulation on ICaand ICl in guinea pig ventricular myocytes to gain insights into possible differences in the regulation of these two currents (Fig. 1). Myocytes were voltage-clamped using the whole-cell configuration of the patch-clamp technique. K+ currents were blocked by internal tetraethylammonium and internal and external cesium. The inward Na+ current was blocked by holding the membrane potential at −50 mV. Myocytes were routinely depolarized from a holding potential of −50 to 0 mV for 200 msec at a frequency of 0.1 sec−1. The depolarization elicited a transient inward current that was completely blocked by Ca2+ channel blockers. ICawas measured as the difference between the peak inward current and the current at the end of the depolarizing pulse. IClwas measured as the steady state current at −50 mV. The current at −50 mV that is stimulated by Iso is ICl under these conditions because the reversal potential (Erev) of this current obtained by voltage ramps was near ECl (data not shown). Because ICa was measured near the Erev for ICl, there was little contamination of ICa by ICl.

    Figure 1
    View larger version:
    Figure 1

    Effect of Iso on ICa (○) and ICl (▪) in guinea pig ventricular myocytes. A, Time course of the effect of Iso; 1 μm was added to the superfusion (bar). Representative current traces are shown for control (A, a) and after Iso (B,b). B, Comparison of onset and washout of the effect of Iso on ICa and ICl. Currents were normalized to the maximum current stimulated by Iso.

    In Fig. 1A, 1 μm Iso increased ICa3.5-fold and increased ICl to 2.2 pA/pF. ICa was stimulated more slowly than ICl. Fig. 1B shows the time course of change of the normalized currents. The time to half-maximum stimulation was 12.0 sec for ICl and 24.6 sec for ICa (Fig. 1B). On washing out Iso, ICa also decreased more slowly than ICl. The time to half-maximum deactivation was 1.6 min for ICl and 3.1 min for ICa. On average, the half-times of activation and inactivation for ICa were 25.8 ± 0.6 sec and 3.6 ± 0.2 min, respectively (five cells), and the half-times of activation and inactivation for ICl were 12.0 ± 0.2 sec and 2.1 ± 0.2 min, respectively (six cells). In Fig. 1B, it seems that ICl in this cell may have washed out with two kinetic components, one fast and one slow. This was not a routine observation.

    Effects of protein phosphatase inhibitors on ICa and ICl.

    To investigate further the phosphorylation reactions controlling these currents, we examined the effects of protein phosphatase inhibitors. Fig. 2A shows the effects of internal perfusion of the myocyte with the protein phosphatase inhibitor microcystin. The bar indicates the period when microcystin was added to the internal solution. On changing the internal solution, there was a ∼3-min lag period before ICa increased. This was partly due to the dead volume of the internal perfusion system. ICaincreased from 2.2 to 11.0 pA/pF in 13 min in response to 10 μm microcystin. However, ICl did not begin to increase until after ICa had nearly reached its maximum (11 min after changing the internal solution); then, ICl increased very slowly. At 18 min after the addition of 10 μm microcystin, ICl was 1.2 pA/pF. Microcystin (10 μm) stimulated both ICa and ICl, but ICa always increased more rapidly than ICl. The subsequent application of Fsk stimulated ICl markedly but had little effect on ICa.

    Figure 2
    View larger version:
    Figure 2

    Effects of the protein phosphatase inhibitor microcystin on ICa and ICl. A, 10 μm microcystin was applied by internal perfusion, and 10 μm Fsk was applied to the superfusion as indicated. Internal perfusion was begun at the time indicated, but ∼3 min was required for the new solution to reach the tip of the pipette. Microcystin increased both ICa (○) and ICl(▪), but ICl increased more slowly than ICa. B, 3 μm microcystin increased ICa a small amount but had no effect on ICl (trace B). The subsequent application of 10 μm Fsk produced a large and rapid increase in both currents (trace C). C, Effect of internal perfusion with 1 μm microcystin. Neither ICanor ICl was stimulated by microcystin alone.

    Fig. 2, B and C, shows the effects of lower concentrations of microcystin. These lower concentrations of microcystin (1 or 3 μm) had no effect on ICl and had only minimal effects on basal ICa. However, subsequent application of 10 μm Fsk produced a large, rapid increase in both currents. Both currents remained partially elevated after Fsk washout. Thus, lower concentrations of microcystin seemed to inhibit a phosphatase responsible for dephosphorylation of sites phosphorylated by PKA but did not significantly stimulate the currents in the absence of PKA activity.

    The very slow stimulation of ICl by microcystin shown in Fig. 2A was in contrast to the rapid stimulation of ICl relative to ICa by Iso in Fig. 1. The relative difference in the kinetics of stimulation of ICa and ICl by Iso and microcystin suggests that there are interesting differences in how phosphorylation regulates these channels.

    Fig. 3 shows the dose-response curves for the effect of microcystin alone on ICa (Fig. 3A) and ICl (Fig. 3B). The effects of microcystin on both ICa and ICl were measured ∼2–5 min after the effect of microcystin on ICa had plateaued. Because the effect of microcystin on ICl is much slower than the effect on ICa, the dose-response curve for ICl was not obtained in a steady state condition and may actually underestimate the steady state effect of microcystin. However, it was not practical to wait until the effect on ICl reached a plateau. Despite this limitation, the data demonstrate that the effect of microcystin was quantitatively less on ICl than it was on ICa over the same time period. Both currents were activated by microcystin at a threshold concentration of ∼3 μm . Data were fitted to the Hill equation (lines). The best-fit parameters were ICa,K½ = 4.6 μm, Imax = 5.5 pA/pF; and ICl,K½ = 7.0 μm, Imax = 1.46 pA/pF. The effects of microcystin on ICa and ICl are compared in Fig. 3C. At 20 μm, microcystin alone was able to stimulate ICa as much as microcystin plus Iso, but in the same time period, the same concentration of microcystin alone was able to stimulate ICl only ∼40% compared with the stimulation of ICl produced by microcystin plus Iso. This suggests that whatever kinases are responsible for the increases in ICa produced by microcystin alone are not as effective in stimulating ICl.

    Figure 3
    View larger version:
    Figure 3

    Dose-response curve for the effect of microcystin on ICa (A) and ICl (B). Data points represent mean (5–13 cells) increase in ICa and IClproduced by a single internal application of microcystin. Data were fitted to the Hill equation (lines). The best-fit parameters were ICa, K½= 4.6 μm, Imax = 5.5 pA/pF; and ICl, K½ = 7.0 μm, Imax = 1.46 pA/pF. C, Microcystin-stimulated current relative to current stimulated in the presence of microcystin and Fsk together. ○, ICa. ▪, ICl. The best-fit parameters were ICa,K½ = 4.8 μm, Imax = 1.27 pA/pF; and ICl,K½ =15.0 μm, Imax = 0.74 pA/pF.

    The ICa stimulated by microcystin had the same biophysical properties as the basal ICa. Fig.4A shows that the shape of the peak current-voltage relationship of ICa was unaffected by microcystin, but the current was increased in amplitude at all potentials. The activation curve and inactivation curve also were not affected (Fig. 4, B and C).

    Figure 4
    View larger version:
    Figure 4

    Effects of microcystin on current-voltage relationship, activation, and inactivation curves of ICa. A, Peak current-voltage relationships before (○) and after (•) addition of 10 μm microcystin. Values are mean of five cells. B, Activation curves. The values were normalized to the maximum ICa. Continuous curves, fits to the Boltzmann relation. The half-maximal activation voltage (½) was −17.5 mV for control and −18.0 mV after the addition of 10 μm microcystin. C, Inactivation curve obtained by a double-pulse protocol before (circo]) and after (•) addition of 10 μm microcystin. The double-pulse protocol consisted of a 200-msec test pulse to 0 mV preceded by various conditioning prepulses of 2-sec duration between −50 and +10 mV separated by a 3-msec repolarization to −80 mV. Values for V½ were −18.2 mV for control and −20.0 mV after addition of 10 μm microcystin.

    The microcystin-stimulated current at −50 mV is carried by Cl−.

    For work described in the preceding paragraphs, we assumed that the current at −50 mV stimulated by high concentrations of microcystin (Fig. 2A) was ICl. To verify this, we measured the current-voltage relationship of the microcystin-induced current. The current-voltage relationship was determined by voltage ramps from −50 to +50 mV at 0.34 V/sec (Fig.5). In these experiments, we used the standard external solution except that 2 mmCoCl2 replaced 1.8 mmCaCl2 to eliminate ICa. Fig. 5A shows current-voltage relations before and after internal perfusion with 10 μm microcystin. The microcystin-stimulated current was obtained by subtracting the before-perfusion value from the after-perfusion value (Fig. 5B). The current-voltage relationship was essentially linear with a small amount of outward rectification. The current reversed at −7.3 mV, which was very close to ECl (−9.6 mV). To verify that the current was a Cl− current, we changed the bath [Cl−]o. The mean current-voltage relations of the microcystin-induced current with 28.6 and 158 mm[Cl−]o were determined as the difference between the average of five records obtained in the absence and presence of microcystin (Fig. 5C). The reversal potential was −7.1 ± 1.9 mV with 158 mm[Cl−]o and shifted to +36.2 ± 3.9 mV with 28.6 mm[Cl−]o. These values were almost identical to the calculated ECl value in each condition (ECl = −9.6 and +34.8 mV in 158 and 28.6 mm[Cl−]o, respectively, with [Cl−]i = 109 mm). The dotted lines indicate the current-voltage relationship calculated from the Goldmann-Hodgkin-Katz equation (10) assuming a simple Cl− conductance. From these data, we conclude that the microcystin-stimulated background current was carried by Cl−. Because intracellular Ca2+ is highly buffered, it is unlikely this is a Ca2+-activated Cl−conductance and thus it is probably the CFTR Cl−channel.

    Effects of microcystin at 36o.

    The results in Fig.2 differ somewhat from those of Hwang et al. (7), who showed that after washout of Fsk in the presence of microcystin, ICl decreased to a level ∼25% of the maximally stimulated current. In contrast, we found that the effect of Fsk on ICl in the presence of 10 μmmicrocystin was nearly irreversible (Fig. 2A). One difference between our experiments and their experiments was that ours were performed at room temperature, whereas theirs were performed at 36°. When we repeated our experiments at 36° (Fig.6, A and B), the stimulatory effects of 10 μm microcystin on ICa and ICl were qualitatively the same as at room temperature. Subsequent exposure to Fsk caused an rapid increase in ICl, but in contrast to the results at room temperature, washout of Fsk was followed by a ∼75% decrease in ICl (Fig. 6, A and B). These results confirm those of Hwang et al. (7) and suggest that the difference between our result and theirs is due to higher phosphatase activity at 36°, which results in a partial dephosphorylation of ICl.

    Figure 6
    View larger version:
    Figure 6

    Inhibitory effects of microcystin and Fsk on ICa and ICl at 36° and room temperature. At 10 μm, microcystin was perfused internally, and 10 μm Fsk was added to the superfusion as indicated. A, Temperature = 36°. The amplitudes of ICa (○) and ICl (▪) are plotted versus time. B, Sample traces from the experiment shown in A. C, Temperature = 22°. Dose-response curve for the effect of calyculin A on ICa. ○, ICa density stimulated by calyculin A alone. □, ICa density in the presence of calyculin and Iso. D, Dose-response curve for the effect of calyculin A on ICl. ○, ICl density stimulated by calyculin A alone. □, ICl density in the presence of calyculin and Iso. Values in C and D are mean of 4–10 cells. ∗, Statistical significance from current in the absence of calyculin at 1% level (ttest).

    Inhibitory effects of microcystin and Fsk on ICa.

    The experiments at 36° revealed a very interesting aspect of ICa regulation that was less evident but also present at room temperature. Although ICa was stimulated by internal perfusion with 10 μm microcystin at 36°, subsequent exposure to Fsk produced a complex response in ICa. Initially, ICa rose quickly, but after ∼30 sec, it began to decline. This decline continued even after Fsk was washed out. These results suggested that at 36°, Fsk may have triggered an event that caused a decrease in Ca2+ channel activity. Additional evidence for this inhibitory effect of Fsk can be seen at room temperature (Fig.2A); ICa in the presence microcystin alone was stable for ∼5 min, but after exposure to Fsk, ICa began to decrease, although ICl did not change.

    To examine this effect in more detail, we examined the effect on ICa and ICl of another protein phosphatase inhibitor, calyculin A. Fig. 6C shows the effects of calyculin A and calyculin A plus Iso on ICa, and Fig. 6D shows the effects on ICl. Iso stimulated ICl to approximately the same degree, regardless of the concentration of calyculin A present. The amplitudes of the Cl− currents in the presence of any concentration of calyculin and Iso were not significantly different than the amplitude in the absence of calyculin (>5%, ttest). In contrast, the stimulation of ICa by Iso decreased as the calyculin A concentration increased over the same range. The amplitude ot the Ca2+ current in the presence of Iso and 150 nm calyculin was significantly different than the amplitude in the absence of calyculin (<1%,t test). These results suggest the possibility that there is an inhibitory phosphorylation that is potentiated by protein phosphatase inhibitors that selectively affects ICa. The fact that ICl is not affected shows that the effect is not a nonspecific effect.

    Effects of protein kinase inhibitors on the response of ICa and ICl to microcystin.

    We hypothesize that microcystin stimulates ICa and ICl because there is a basally active protein kinase whose activity is counteracted by a basally active protein phosphatase. When the phosphatase is inhibited by microcystin, the phosphoprotein can accumulate and the current amplitude increases. In our studies on frog cardiomyocytes, we showed that the effect of microcystin required ATP and was blocked by protein kinase inhibitors (6, 11). To test whether the same mechanism could explain the effect of microcystin in guinea pig, we examined the ability of staurosporine to block the effects of microcystin (Fig.7). Staurosporine, which is a nonspecific protein kinase inhibitor (12, 13), did block the effect of microcystin on ICa and ICl. Staurosporine was applied extracellularly at concentrations of 100–300 nm before internal perfusion with 20 μmmicrocystin was begun. At 300 nm, staurosporine decreased basal ICa and nearly completely inhibited the increase in ICa and IClproduced by microcystin (Fig. 7A). Even though 300 nmstaurosporine nearly completely blocked the response of ICa and ICl to microcystin, subsequent application of Fsk produced large increases in both currents. Fig. 7B shows the dose-response curve for the effect of staurosporine on the microcystin-stimulated current. The EC50 values for inhibition of the effect of staurosporine on ICa and ICl were 171.5 and 161 nm, respectively. On average, in the presence of 300 nmstaurosporine, microcystin-stimulated ICa and ICl were 0.48 ± 0.13 and 0.46 ± 0.22 pA/pF, respectively. In comparison, ICa and ICl values in the presence of 300 nmstaurosporine, 20 μm microcystin, and 10 μmFsk were 5.5 ± 0.79 and 3.4 ± 0.43 pA/pF, respectively. These results suggest that staurosporine inhibits a protein kinase that is responsible for stimulation of ICa and ICl by microcystin but that staurosporine at these concentrations has only a small effect on PKA.

    Figure 7
    View larger version:
    Figure 7

    Effect of the protein kinase inhibitor staurosporine on the response of ICa and ICl to microcystin. ○, ICa. ▪, ICl. A, 300 nm staurosporine and 10 μm Fsk were applied by superfusion, and 20 μm microcystin was applied by internal perfusion during the times indicated. Staurosporine decreased basal ICa and inhibited the effect of microcystin on ICa and ICl. Subsequent application of Fsk produced a large increase in ICa and ICl even in the presence of staurosporine. B, Dose-response curve for the effect of staurosporine on microcystin-stimulated ICa and ICl. Data were fitted to the power logistic equation (lines). EC50 values were 171.5 nm for ICa and 161 nm for ICl. Response of ICa (▵) and ICl(▾) to 10 μm Fsk in the presence of 300 nmstaurosporine and 20 μm microcystin (five cells).

    Another interpretation that must be entertained is that staurosporine inhibits the action of microcystin nonspecifically via a mechanism independent of protein kinase inhibition. Although we cannot absolutely exclude this possibility, there are a number of reasons why this seems unlikely. First, in frog myocytes, in which microcystin produces a stimulation of ICa and an inhibition of the delayed rectifier K+ current IK, only the stimulation of ICa is inhibited by staurosporine (6). Therefore, the action of staurosporine is not nonspecific. Second, it seems clear that a kinase is involved in the action of microcystin on ICa because the effect requires ATP and is blocked not only by staurosporine but also by other kinase inhibitors such as K252a and H7 (6). Furthermore, the inhibitory effect of staurosporine is only observed if it is applied before the stimulatory effect of microcystin has occurred. If staurosporine is applied after microcystin has already stimulated the current, staurosporine is unable to inhibit the current. This behavior is consistent with a mechanism involving microcystin inhibiting a phosphatase and staurosporine inhibiting a kinase because one would expect staurosporine to have no effect once phosphorylation had taken place in the presence of an inhibitor of dephosphorylation (microcystin). It is difficult to propose a nonspecific mechanism of staurosporine action that has this behavior. Because a kinase is clearly involved in the response to microcystin and because the effect of microcystin is blocked by staurosporine, a known kinase inhibitor, we favor the simplest interpretation that staurosporine blocks the effect of microcystin by inhibition of a kinase.

    Staurosporine also reduced basal ICa. On average, 300 nm staurosporine reduced basal ICa by 29.5 ± 10.1% (six cells). This decrease did not seem to be rundown because the effect of staurosporine was rapid. This suggests that the basal amplitude of ICa may be determined by a kinase that is basally active and inhibited by staurosporine.

    PKA inhibition has no effect on the response of ICa and ICl to microcystin.

    To determine which protein kinases might be responsible for the stimulation of these currents by microcystin, we examined the effects of the competitive PKA inhibitor Rp-cAMPS on the responses of ICa and ICl to 10 μm microcystin (Fig.8A). In this experiment, the cells were first perfused internally with 2 mm Rp-cAMPS, which had very little effect on basal ICa. After ∼7-min internal perfusion with Rp-cAMPS, neither ICa nor ICl responded to 1 μm Iso, showing that PKA had been inhibited. However, the subsequent addition of 10 μm microcystin to the internal perfusion produced a large stimulation of both ICa and ICl. To be certain that PKA had been completely inhibited, we also performed this experiment in a different order (Fig. 8B). In this cell, Rp-cAMPS completely blocked PKA because Fsk had no effect on either ICa or ICl when added after microcystin perfusion, but microcystin was still capable of increasing both currents. The mean values for 10 μmmicrocystin-stimulated ICa and ICl in the presence of Rp-cAMPS were 6.0 ± 0.4 and 1.1 ± 0.1 pA/pF, respectively (six cells). The mean values for 10 μm microcystin-stimulated ICa and ICl without Rp-cAMPS were 4.9 ± 0.5 and 1.0 ± 0.12 pA/pF, respectively (15 cells). These results show that the effects of microcystin on ICa or IClwere not mediated by phosphorylation catalyzed by PKA.

    Figure 8
    View larger version:
    Figure 8

    Effect of the PKA inhibitor Rp-cAMPS on the response of ICa and ICl to 10 μm microcystin and 1 μm Iso (A) or 10 μm Fsk (B). ○, ICa. ▪, ICl. Cells were first perfused internally with 2 mmRp-cAMPS, which had no effect on basal ICa. A, Response to Iso and microcystin. B, Response to microcystin and Fsk.

    PKC inhibition has no effect on the response of ICa and ICl to microcystin.

    Staurosporine is a nonselective protein kinase inhibitor, and it inhibits PKC in vitro at submicromolar concentrations (13). This suggests the possibility that the basally active protein kinase that stimulates ICa and ICl in the presence of microcystin is PKC. PKC is known to activate ICa in guinea pig ventricular myocytes (14), but it is controversial whether PKC regulates ICl(3). Fig. 9A shows the effect of the PKC activator TPA (0.1 μm) on ICa and ICl. ICa began to increase within 1 min after exposure to TPA and reached 11.3 pA/pF. ICl began to increase more slowly than ICa and reached a density of 2.3 pA/pF. Thus, it seems that both currents may be regulated by PKC. To determine whether the effect of microcystin could be explained by PKC-dependent phosphorylation, we examined the effects of a highly selective pseudosubstrate PKC inhibitor.

    Figure 9
    View larger version:
    Figure 9

    Regulation of ICa and IClby PKC and microcystin. ○, ICa. ▪, ICl. A,. Effects of the PKC activator TPA on ICa and ICl. At 0.1 μm, TPA was applied to the bath solution during the time indicated (bar). ICa began to increase within 1 min after exposure of TPA. ICl began to increase slower than ICa. B, Effects of a PKC pseudosubstrate inhibitor on the response of ICa and ICl to microcystin; 100 μm myr-PKC(19−31) was applied internally with 2 mm Rp-cAMPS. After a 6-min perfusion with myr-PKC(19−31) and Rp-cAMPS, 0.1 μm TPA had no effect on basal ICa and ICl. Subsequent internal perfusion with 10 μmmicrocystin increased both ICa and ICl to 7.1 and 1.8 pA/pF, respectively.

    For these experiments, we used the myristoylated derivative of the peptide pseudosubstrate inhibitor PKC(19–31); the myristoylated derivative has been reported to be a more effective inhibitor of PKC because it is targeted to the membrane (15). In Fig.9B, 100 μm myr-PKC(19–31) was applied internally with 2 mm Rp-cAMPS. After a 6-min perfusion with myr-PKC(19–31) and Rp-cAMPS, 0.1 μm TPA had no effect on ICa or ICl. However, 10 μm microcystin increased both ICaand ICl to 7.1 and 1.8 pA/pF, respectively. These results show that stimulation of ICa and ICl by microcystin is not mediated by PKC.

    Previous SectionNext Section

    Discussion

    ICa and ICl are regulated independently by phosphorylation.

    These data show that although both ICa and ICl are regulated by PKA phosphorylation, their regulation is not necessarily coordinate. For example, the increases in amplitude of ICaand ICl produced by Iso occur with different kinetics. ICl increases more rapidly than ICa in response to stimulation by Iso; the washout of the Iso effect is also faster. If we assume that the rate-limiting step regulating the amplitudes of ICa and ICl is a phosphorylation reaction, D ⇄ P, then the rate of increase in the current will be proportional to α + β and deactivation will be proportional to β, where α is the forward rate constant and β is the backward rate constant. Because both the stimulation and washout of the Iso effect were faster for ICl, we conclude that the dephosphorylation reaction for ICl is faster than that for ICa. We do not currently have any insights into why dephosphorylation of ICl may be faster than ICa; possibilities include different phosphatase isoforms responsible for dephosphorylation of the two channels or differences in the rate of dephosphorylation of the different substrates by the same phosphatase.

    ICa and ICl are regulated by PKA and another protein kinase.

    The observation that ICl was more rapidly dephosphorylated than ICa suggested that application of a protein phosphatase inhibitor might result in a larger basal stimulation of ICl than ICa. However, we found that ICl increased much less and more slowly than ICa in response to protein phosphatase inhibitors. This difference in the kinetics of response of the two currents to Iso and microcystin was explained by the fact that the increase in both basal currents produced by microcystin was not caused by PKA but by another, as yet unidentified, protein kinase. The fact that the effects of microcystin are blocked by low concentrations of the nonselective protein kinase inhibitor staurosporine, however, argues that the effect of microcystin is mediated through a phosphorylation reaction. This protein kinase is clearly not PKA because it is not inhibited by PKA inhibitors, which in the same cell do inhibit the effect of Iso or Fsk. Likewise, the kinase is not PKC because the peptide inhibitors of PKC that inhibit the effects of TPA in the same cell do not inhibit the effects of microcystin. Finally, it is unlikely that the kinase is a Ca2+-dependent kinase because the cells were perfused internally with 10 mm EGTA, which should inhibit the activation of Ca2+-dependent protein kinases. This novel protein kinase is very similar to one we described in frog ventricular myocytes and have termed PKX (6, 11).

    We believe that the microcystin-stimulated current at −50 mV is the CFTR Cl− current (16-18) for the following reasons. (1) The current at −50 mV that is stimulated by microcystin is a Cl− current because it has a reversal potential near ECl and the reversal potential changes as predicted with changes in [Cl−]o. Furthermore, the current-voltage relationship is the same as reported for the cAMP-activated Cl− current. (2) The current does not resemble other Cl− currents that have been described in cardiac muscle, including a stretch-activated Cl− current (19, 20), a Ca2+-activated Cl− current (21, 22), and a PKC-activated Cl− current (23,24). Collier and Hume (25) recently reported that this PKC-activated Cl− current was the CFTR Cl− current in guinea pig ventricular myocytes. It is unlikely that the current is a Ca2+-activated Cl− current because intracellular Ca2+ is highly buffered with EGTA. Furthermore, the fact the microcystin-stimulated current is time independent and requires phosphorylation to be activated suggests it is the CFTR Cl− current.

    Differences in the rates of phosphorylation/dephosphorylation by PKA and PKX could be due to differences in the association of kinases and phosphatases with Cl− and Ca2+ channels. For example, PKX might be more closely associated with Ca2+ channels than Cl− channels such that Ca2+ channels are phosphorylated preferentially. This possibility is made more attractive by recent studies that have identified several “scaffold” proteins that bind and target a variety of signaling enzymes to appropriate locations in the cell (26,27). Recently, it was shown that voltage-dependent potentiation of L-type Ca2+ channels requires a cAMP-anchoring protein (28) and that L-type Ca2+ channels are associated with a 15-kDa cAMP-anchoring protein (29). Thus, differential regulation of different ion channels by protein kinases and phosphatases may be affected by differential localization of the kinases and phosphatases to the immediate neighborhood of the channel.

    Physiological role of PKX.

    We hypothesize that the role of PKX in ventricular myocytes may be to regulate the basal level of ICa. Our reasons for suggesting this hypothesis are discussed below. The basal level of ICa is not determined by PKA-dependent phosphorylation because we find that internal perfusion of guinea pig myocytes with the PKA inhibitor Rp-cAMPS did not have a significant effect on basal ICa (see, for example, Fig. 8A). Ono and Fozzard (30) reported that the phosphatase inhibitor okadaic acid slowed rundown of Ca2+ channel activity in excised inside-out patches. They suggested that dephosphorylation was an important component of rundown and that phosphorylation was needed for channel-opening activity under basal conditions. They speculated that the intact cell may have protein kinases other than PKA that could maintain some level of phosphorylation. This is consistent with our results, and we conclude PKX may be important for maintaining basal phosphorylation of ICa in guinea pig ventricular myocytes. In support of this suggestion is the observation that 300 nm staurosporine reduced basal ICa, which may reflect basal activation of ICa by PKX.

    Hescheler et al. (31, 32) have also reported that cell dialysis with protein phosphatase 1 inhibitor or okadaic acid stimulated basal ICa and suggested that there was a high activity of intracellular protein kinase even in the absence of agonist that was normally counteracted by a high level of protein phosphatase activity.

    The role of PKX in regulating ICl is less clear. ICl is stimulated much more slowly and to a much lesser extent than ICa. Thus, it is not clear that this phosphorylation is physiologically relevant. The observation that ICl is activated slowly by phosphatase inhibitors suggests that the Cl− channel is also a substrate for PKX but that either the Cl−channel is a relatively poor substrate for PKX or protein phosphatases that are insensitive to microcystin keep the accumulation of the phosphorylated state low. Cheng et al. (33) detected basal levels of phosphorylation in CFTR Cl− channel and suggest that basal phosphorylation sites do not represent PKA sites in CFTR Cl− channel. In contrast, Nakashima and Ono (34) observed that the phosphatase inhibitor okadaic acid increased ICl, but they speculated that this was due to basal activity of PKA.

    Inhibitory effects of PKA stimulation in the presence of protein phosphatase inhibitors.

    Another novel finding of these studies is that Fsk or Iso in the presence of microcystin or calyculin A seemed to have a dual effect on ICa; initially, Fsk or Iso stimulated ICa, but then it initiated a run-down of ICa. This decrease in ICa was not paralleled by any change in ICl, suggesting that run-down of ICa was not due to deterioration of the cell or to activation of a nonselective microcystin-insensitive phosphatase. The effect was observed with both Fsk and Iso, suggesting that the effect was not due to a pharmacological effect of Fsk but rather to activation of the cAMP cascade. This run-down phenomenon was much more pronounced at 36°. The decrease in the amplitude was accompanied by increased inactivation of the current, supporting the idea that the effect was specific.

    In conclusion, we found that ICa and ICl are stimulated by phosphatase inhibitors in the absence of β-adrenergic stimulation. The conclusion that this stimulatory effect is mediated by a basally active kinase is supported by the observation that this effect is inhibited by a protein kinase inhibitor. The kinase is not PKA nor PKC because it is not inhibited by PKA or PKC inhibitors. We speculate that this kinase, which we term PKX, contributes to set the basal level of ICabut plays a lesser role in regulation of the CFTR Cl− channel.

    Previous SectionNext Section

    Acknowledgments

    We thank A. Rinderknecht and Drs. L. Quarmby, A. Ivanov, K. Machaca, S. Mierergerd, and K. Uchida for comments on the manuscript.

    Previous SectionNext Section

    Footnotes

    • Send reprint requests to: Dr. H. Criss Hartzell, Department of Cell Biology, Emory University School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322-3030. E-mail: criss{at}anatomy.emory.edu

    • This work was supported by National Institutes of Health Grant HL21195.

    • Abbreviations:
      PKA
      protein kinase A
      CFTR
      cystic fibrosis transmembrane conductance regulator
      ECl
      Cl− equilibrium potential
      Erev
      reversal potential
      Fsk
      forskolin
      ICa
      L-type Ca2pluscurrent
      ICl
      cAMP-activated Cl− current
      Iso
      isoproterenol
      myr-PKC(19–31)
      myristoylated derivative of the pseudosubstrate protein kinase C inhibitor
      PKC
      protein kinase C
      PKX
      protein kinase X
      TPA
      12-O-tetradecanoylphorbol-13-acetate
      EGTA
      ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      Rp-cAMPS
      adenosine 3′-5′-cyclic phosphorothioate
      • Received April 8, 1997.
      • Accepted June 18, 1997.
    Previous Section
     

    References

    1. ↵
      1. Hartzell H. C.
      (1985) Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systems. Prog. Biophys. Mol. Biol. 52:165–247.
      CrossRef
    2. ↵
      1. McDonald T. F.,
      2. Pelzer S.,
      3. Trautwein W.,
      4. Pelzer D. J.
      (1994) Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol. Rev. 74:365–507.
      FREE Full Text
    3. ↵
      1. Gadsby D. C.,
      2. Nagel G.,
      3. Hwang T. C.
      (1995) The CFTR chloride channel of mammalian heart. Annu. Rev. Physiol. 57:387–416.
      CrossRefMedlineWeb of Science
    4. ↵
      1. Tsien R. W.,
      2. Bean B. P.,
      3. Hess P.,
      4. Lansman J. B.,
      5. Nilius B.,
      6. Nowycky M. C.
      (1986) Mechanism of calcium channel modulation by β-adrenergic agents and dihydropyridine calcium agonists. J. Mol. Cell Cardiol. 18:691–710.
      MedlineWeb of Science
    5. ↵
      1. Ono K.,
      2. Fozzard H. A.
      (1993) Two phosphatase sites on the Ca2+ channel affecting different kinetic functions. J. Physiol. (Lond.) 470:73–84.
      Abstract/FREE Full Text
    6. ↵
      1. Hartzell H. C.,
      2. Hirayama Y.,
      3. Petit-Jacques J.
      (1995) Effects of protein phosphatase and kinase inhibitors on the cardiac L-type Ca current suggest two sites are phosphorylated by protein kinase A and another protein kinase. J. Gen. Physiol. 106:393–414.
      Abstract/FREE Full Text
    7. ↵
      1. Hwang T. C.,
      2. Horie M.,
      3. Gadsby D. C.
      (1993) Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl− conductance in mammalian heart. J. Gen. Physiol. 101:629–650.
      Abstract/FREE Full Text
    8. ↵
      1. Hartzell H. C.,
      2. Rinderknecht A.
      (1996) Calphostin C, a widely used protein kinase C inhibitor, directly and potently blocks L-type Ca channels. Am. J. Physiol. 270:C1293–C1299.
      Abstract/FREE Full Text
    9. ↵
      1. Matsuoka S.,
      2. Ehara T.,
      3. Noma A.
      (1990) Chloride-sensitive nature of the adrenaline-induced current in guinea-pig cardiac myocytes. J. Physiol. 425:579–598.
      Abstract/FREE Full Text
    10. ↵
      1. Hille B.
      (1992) Ionic Channels of Excitable Membranes (Sinauer Associates, Sunderland, MA), 2nd ed. p 341.
    11. ↵
      1. Frace A. M.,
      2. Hartzell H. C.
      (1993) Opposite effects of phosphatase inhibitors on L-type calcium and delayed rectifier currents in frog cardiac myocytes. J. Physiol. (Lond.) 472:305–326.
      Abstract/FREE Full Text
    12. ↵
      1. Tamaoki T.,
      2. Nomoto H.,
      3. Takahashi I.,
      4. Kato Y.,
      5. Morimoto M.,
      6. Tomita F.
      (1986) Staurosporine, a potent inhibitor or phospholipid/Ca dependent protein kinase. Biochem. Biophys. Res. Commun. 135:397–402.
      CrossRefMedlineWeb of Science
    13. ↵
      1. Rhegg U. T.,
      2. Burgess G. M.
      (1989) Staurosporin, K-252, and UCN-01: potent but nonspecific inhibitors of protein kinases. Trends Pharmacol Sci. 10:18–22.
      CrossRefMedline
    14. ↵
      1. Lacerda A. E.,
      2. Rampe D.,
      3. Brown A. M.
      (1988) Effects of protein kinase C activators on cardiac Ca2+ channels. Nature (Lond.) 335:249–251.
      CrossRefMedline
    15. ↵
      1. Eicholtz T.,
      2. deBont D. B.,
      3. deWidt J.,
      4. Liskamp R.-M.,
      5. Ploegh H.-L.
      (1993) A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor. J. Biol. Chem. 268:1982–1986.
      Abstract/FREE Full Text
    16. ↵
      1. Bahinski A.,
      2. Nairn A. C.,
      3. Greengard P.,
      4. Gadsby D. C.
      (1989) Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes. Nature (Lond.) 340:718–721.
      CrossRefMedline
    17. ↵
      1. Harvey R. D.,
      2. Hume J. R.
      (1989) Autonomic regulation of a chloride current in heart. Science (Washington D. C.) 244:983–985.
      Abstract/FREE Full Text
    18. ↵
      1. Ehara T.,
      2. Ishihara K.
      (1990) Anion channels activated by adrenaline in cardiac myocytes. Nature (Lond.) 347:284–286.
      CrossRefMedline
    19. ↵
      1. Hagiwara N.,
      2. Matsuda H.,
      3. Shoda M.,
      4. Irisawa H.
      (1992) Stretch-activated anion currents of rabbit cardiac myocytes. J. Physiol. (Lond.) 456:285–302.
      Abstract/FREE Full Text
    20. ↵
      1. Sorota S.
      (1992) Swelling-induced chloride-sensitive current in canine atrial cells revealed by whole-cell patch-clamp method. Circ. Res. 70:679–687.
      Abstract/FREE Full Text
    21. ↵
      1. Sipido K.,
      2. Callewaert G.,
      3. Carmeliet E.
      (1993) [Ca2+]i transients and [Ca2+]i-dependent chloride current in single Purkinje cells from rabbit heart. J. Physiol. (Lond.) 468:641–667.
      Abstract/FREE Full Text
    22. ↵
      1. Kawano S.,
      2. Hirayama Y.,
      3. Hiraoka M.
      (1995) Activation mechanism of Ca2+-sensitive transient outward current in rabbit ventricular myocytes. J. Physiol. (Lond.) 486:593–604.
      Abstract/FREE Full Text
    23. ↵
      1. Walsh K. B.,
      2. Long K. J.
      (1994) Properties of a protein kinase C-activated chloride current in guinea pig ventricular myocytes. Circ. Res. 74:121–129.
      Abstract/FREE Full Text
    24. ↵
      1. Zhang K.,
      2. Barrington P. L.,
      3. Martin R. L.,
      4. Ten Eick R. E.
      (1994) Protein kinase-dependent Cl− currents in feline ventricular myocytes. Circ. Res. 75:133–143.
      Abstract/FREE Full Text
    25. ↵
      1. Collier M. L.,
      2. Hume J. R.
      (1995) Unitary chloride channels activated by protein kinase C in guinea pig ventricular myocytes. Circ. Res. 76:317–324.
      Abstract/FREE Full Text
    26. ↵
      1. Faux M. C.,
      2. Scott J. D.
      (1996) Molecular glue: kinase anchoring and scaffold proteins. Cell 85:9–12.
      CrossRefMedlineWeb of Science
    27. ↵
      1. Klauck T. M.,
      2. Faux M. C.,
      3. Labudda K.,
      4. Langeberg L. K.,
      5. Jaken S.,
      6. Scott J. D.
      (1996) Coordination of three signalling enzymes by AKAP79, a mammalian scaffold protein. Science (Washington D. C.) 271:1589–1592.
      Abstract
    28. ↵
      1. Johnson B. D.,
      2. Scheuer T.,
      3. Catterall W. A.
      (1994) Voltage-dependent potentiation of L-type Ca2+ channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 91:11492–11496.
      Abstract/FREE Full Text
    29. ↵
      1. Gray P. C.,
      2. Tibbs V. C.,
      3. Catterall W. A.,
      4. Murphy B. J.
      (1997) Identification of a 15-kD cAMP-dependent protein kinase-anchoring protein associated with skeletal muscle L-type calcium channels. J. Biol. Chem. 272:6297–6302.
      Abstract/FREE Full Text
    30. ↵
      1. Ono K.,
      2. Fozzard H. A.
      (1992) Phosphorylation restores activity of L-type calcium channels after rundown in inside-out patches from rabbit cardiac cells. J. Physiol. (Lond.) 454:673–688.
      Abstract/FREE Full Text
    31. ↵
      1. Hescheler J.,
      2. Kameyama M.,
      3. Trautwein W.,
      4. Mieskes G.,
      5. Söling H. D.
      (1987) Regulation of the cardiac calcium channel by protein phosphatases. Eur. J. Biochem. 165:261–266.
      MedlineWeb of Science
    32. ↵
      1. Hescheler J.,
      2. Mieskes G.,
      3. Rhegg J. C.,
      4. Takai A.,
      5. Trautwein W.
      (1988) Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes. Pflueg. Arch. Eur. J. Physiol. 412:248–252.
      CrossRefMedlineWeb of Science
    33. ↵
      1. Cheng S. H.,
      2. Rich D. P.,
      3. Marshall J.,
      4. Gregory R. J.,
      5. Welsh M. J.,
      6. Smith A. E.
      (1991) Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66:1027–1036.
      CrossRefMedlineWeb of Science
    34. ↵
      1. Nakashima Y.,
      2. Ono K.
      (1994) Rate-limiting steps in activation of cardiac Cl− current revealed by photolytic application of cAMP. Am. J. Physiol. 267:H1514–H1522.
      Abstract/FREE Full Text
    « Previous | Next Article »Table of Contents

    This Article

    1. Molecular Pharmacology October 1, 1997 vol. 52 no. 4 725-734
    1. AbstractFree
    2. » Full Text
    3. Full Text (PDF)

    Classifications

    Responses

    1. Submit a response
    2. No responses published

    Navigate This Article

    Current Issue

    1. November 2009, 76 (5)
    1. Current Issue
    1. Alert me to new issues of Molecular Pharmacology