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(Circulation Research. 1998;83:995-1002.)
© 1998 American Heart Association, Inc.

Original Contributions

Independent and Exclusive Modulation of Cardiac Delayed Rectifying K⁺ Current by Protein Kinase C and Protein Kinase A

C. Frederick Lo, , Randal Numann

From Wyeth-Ayerst Research, Princeton, NJ.

Correspondence to Dr Randal Numann, Wyeth-Ayerst Research, CN8000, Princeton, NJ 08543-8000. E-mail numannr{at}war.wyeth.com

	Abstract

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Abstract—Expression of minK in Xenopus oocytes results in a current similar to the cardiac slow delayed rectifying K⁺ (I_Ks) current. Modulation of the I_Ks current in cardiac myocytes has been studied extensively because of its role in shaping the cardiac action potential. The human and cat minK cDNA have been cloned, but their regulation by protein kinases has not been characterized. We report here on the complex modulation of human and cat I_Ks currents by protein kinase C (PKC) and protein kinase A (PKA). Activation of PKC by phorbol ester (100 nmol/L phorbol 12,13-didecanoate [PDD]) produces an increase in I_Ks current that peaks after 20 minutes and then subsequently decreases to 50% of the control level after 1 hour. PKA activation only produces a sustained increase in I_Ks current. Interestingly, premodulation by PKC prevents I_Ks current modulation by PKA, and PKC has no effect on I_Ks current after potentiation by PKA. This shows that the I_Ks current is modulated by PKC and PKA in a mutually exclusive manner and suggests that multiple interacting phosphorylation sites are involved. Activation of PKC by diacylglycerol analogues only produces a slow decrease in I_Ks current. The biphasic effects of PKC on I_Ks current activated by PDD can also be separated by dose and duration. Low doses of PDD (5 nmol/L) or brief applications (5 minutes) of 100 nmol/L PDD only produces I_Ks current activation. These data suggest that there are at least 2 independent PKC phosphorylation sites in the minK-KvLQT1 channel. Additionally, long-term activation of PKC strongly attenuates the I_Ks current expression even when the corresponding changes in capacitance are taken into account.

Key Words: minK current • I_Ks • phosphorylation • protein kinase C • protein kinase A • Xenopus oocyte

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Delayed rectifying K⁺ channels are important in determining the shape and repolarization of cardiac action potentials. One component of the cardiac delay rectifier is termed the K⁺ (I_Ks) current. Recent data strongly suggest that I_Ks is composed of 2 molecular entities: KvLQT1 and minK.^{1 2} Mutations in both components are responsible for the most common forms of inherited long-QT syndrome in humans.^{1 2 3} I_Ks plays an important role in cardiac electrophysiology and its modulation should be equally important, particularly in determining the cardiac action potential duration. I_Ks is modulated in the heart by ß-adrenergic receptors through a cAMP pathway⁴ and by -adrenergic receptors by the PKC pathway.⁵ There is also evidence that Ca²⁺, released intracellularly by IP₃ after activation of either -adrenergic or M3 muscarinic receptors, may modulate I_Ks.⁶ I_Ks is also found in the inner ear, and mutations in minK are found in certain forms of congenital deafness.⁷

When RNA for minK is injected into Xenopus oocytes, the resulting outward current has most of the characteristics of the I_Ks current. This includes a positive midpoint of activation, slow activation and deactivation kinetics, and sensitivity to various blockers such as tetraethylammonium chloride (TEA), Ba²⁺, and azimilide.^{8 9 10} It was speculated initially that minK may form homomeric ion channels^{11 12} ; however, it is now known that the I_Ks current in oocytes is the functional expression of the exogenous minK together with the endogenous KvLQT1 in frogs.²

The complementary DNA of minK has been cloned from many species, including mouse,¹³ rat,⁸ guinea pig,¹⁴ cat,¹⁵ rabbit,¹⁶ and human.¹⁷ I_Ks current, expressed in oocytes from all of these species, was upregulated by PKA activation.¹⁸ Mouse and rat I_Ks currents were decreased by PKC activation,¹³ whereas guinea pig I_Ks current was activated by PKC.¹⁴ In mouse and rat minK there is a putative PKC phosphorylation site at serine 102. Amino acid sequence comparison reveals that guinea pig minK protein has an asparagine residue at the same position. Mutating the guinea pig asparagine into serine creates a mutant guinea pig I_Ks current that is downregulated by PKC.¹⁴ On the other hand, mutating the mouse serine 102 and neighboring residues produces a mutant mouse I_Ks current that is upregulated by PKC.¹⁹ These results suggest that position 102 in the minK protein is critical in determining the effect of PKC. Both human and cat minK have a serine residue at position 102. It could be predicted, therefore, that PKC would downregulate I_Ks currents from human and cat minK. However, we report here that I_Ks currents from human and cat minK are both up- and downregulated by PKC activation. This biphasic effect depends on the dose, duration, and method of PKC activation. We also observe that long-term activation of PKC leads to irreversible downregulation of I_Ks current. In addition, we report that I_Ks current is resistant to potentiation by PKA after prior modulation by PKC, and conversely that PKC modulation will not occur after PKA activation. Our results suggest that PKC and PKA play an important role in modulating I_Ks current and that their modulatory effects are mutually exclusive.

	Materials and Methods

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Oocyte Expression
Adult female Xenopus laevis (Xenopus 1; Ann Arbor, MI) were anesthetized with 0.3% tricaine (Sigma Chemical), and ovarian lobes were removed. The follicular layers were digested by incubation with 2 mg/mL collagenase (type 1A; Sigma Chemical) in OR2 solution (in mmol/L: NaCl 82.5, KCl 2, MgCl₂ 1, HEPES 5; pH 7.4) for 2 hours at room temperature. After digestion, healthy stage V–VI oocytes were manually selected based on size and uniformity of color. Oocytes were cultured in a modified Barth's medium (in mmol/L: NaCl 88, KCl 1, NaHCO₃ 2.4, CaCl₂ 0.4, Ca(NO₃)₂ 0.3, MgSO₄ 0.8, HEPES 20; 5% fetal bovine serum and 150 µg/mL gentamycin) at 15°C to 18°C. cRNA was dissolved in diethylpyrocarbonate H₂O in a concentration of 1 µg/µL. One day after oocytes isolation, human minK, cat minK, or HERG cRNA (20 to 50 ng) were injected by an automatic injector (Drummond Scientific). Oocytes were studied 2 to 12 days after RNA injection.

Electrophysiology
Currents were measured with the conventional 2-microelectrode voltage-clamp technique¹⁵ with a Turbo Tec-10c amplifier (NPI Electronic). Electrodes were fabricated from borosilicate glass (World Precision Instrument) by a Flaming/Brown micropipette puller (Sutter Instrument). The recording electrodes were filled with 3 mol/L KCl, and the typical resistance was 1 M. Oocytes were continuously perfused with ND96 (in mmol/L: NaCl 96, KCl 2, MgCl₂ 1, CaCl₂ 1.8, HEPES 5; pH 7.4) at room temperature (23°C to 25°C). Holding potential was -80 mV in all experiments. Currents were acquired at 1 kHz and filtered at 400 Hz for presentation. Pulse/Pulsefit software (version 8.01, HEKA Electronic) was used for data acquisition and analysis and a P/2 protocol was used for leakage subtraction. The voltage-clamp protocol for measuring I_Ks currents was as follows: a 5-second depolarizing pulse to 30 mV was used to activate the current, followed by a 5-second repolarizing pulse to -70 mV for tail current measurement. I_Ks current amplitudes were measured at the end of the 5-second pulse at 30 mV. This standard voltage protocol was repeated every 30 to 60 seconds. Current-voltage (I-V) relationships were constructed by measuring the tail current amplitudes (at -70 mV) after 5-second depolarizing pulses to various potentials. I-V curves were then fitted to a Boltzmann equation (I/I_max=1/(1+exp^{-(V–V1/2)/k}) with a Levenberg-Marquardt algorithm. Pooled data are reported as mean±SEM. Data were compared with the paired Student t test, and a P<0.05 value was considered statistically significant.

Solutions and Compound Administration
All solutions were added continuously with a perfusion pump at a flow rate of 1 to 2 mL/min. Preliminary experiments with 20 mmol/L K⁺-supplemented ND96 showed that solution change was completed within 2 to 3 minutes. Phorbol 12,13-didecanoate (PDD), 4--phorbol 12,13-didecanoate (4-PDD), and staurosporine were purchased from Sigma. 1,2-Dioctanoyl-sn-glycerol (DOG), 1-oleoyl-2-acetyl-sn-glycerol (OAG), forskolin, chelerythrine, and calphostin C were purchased from Calbiochem. 3-Isobutyl-1-methylxanthine (IBMX) was purchased from Aldrich Chemicals. All compounds were prepared as 200x to 1000x stocks by dissolving in DMSO. The final DMSO concentration in ND96 did not exceed 0.5%, and DMSO alone had no effect on minK or HERG currents. Pseudosubstrate peptide inhibitors of PKA (PKAI_5–24) and PKC (PKCI_19–36) were purchased from Peninsula Laboratories. The kinase inhibitory peptides were dissolved in sterile water and injected into oocytes at a concentration of 250 µmol/L for PKCI, and 120 to 250 µmol/L for PKAI, assuming the volume of oocyte to be 1 µL.²⁰ Oocytes were allowed to recover for 8 to 12 hours before experiments. I_Ks current from oocytes injected with cat minK is referred to as I_Ks (cminK) current and I_Ks (hmink) current for oocytes injected with human minK.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of human I_Ks (hminK) or cat I_Ks (cminK) cRNA in Xenopus oocytes elicited an outwardly rectifying K⁺ current (Figure 1A inset). This I_Ks current was activated at potentials more positive than -40 mV, and the activation time course was slow. I-V relationships could be fitted by a Boltzmann equation with a midpoint of activation (V_1/2) of 12.3±1.9 mV for I_Ks (hminK) current (n=50). I_Ks current could be inhibited by azimilide, with an IC₅₀ of 4±0.7 µmol/L (n=16). Similar results were found for I_Ks (cminK) current.¹⁵ The I-V relationships and pharmacology of both human and cat I_Ks (minK) currents were similar to that of other cloned minK currents such a mouse,¹³ rat,⁸ rabbit,¹⁶ and guinea pig,¹⁴ as well as the native cardiac I_Ks current.^{16 21}

View larger version (17K): [in this window] [in a new window]   Figure 1. Biphasic modulation of IKs current by PDD. A, Representative experiment showing the biphasic response of IKs (hminK) current to 100 nmol/L PDD. Currents were measured every 30 seconds at the end of 5-second depolarization steps to 30 mV and plotted against time. Inset shows current traces at times indicated. Scale bars=500 nA and 2 seconds. B, Normalized I-V relationships obtained from 6 oocytes in experiments similar to panel A. I-V curves were constructed from tail current amplitudes measured at -70 mV after a test pulse from -50 to 60 mV and normalized to the maximal control current (I=1, ). I-V curves from the same oocytes were measured 20 minutes () and 60 minutes () after 100 nmol/L PDD application and scaled to the normalized control current. Lines indicate Boltzmann fits with voltage of half-activation (V1/2) of 7.6±0.7 mV for control (), 10.95±1 mV 20 minutes after PDD (), and 12.6±1 mV 60 minutes after PDD (); slope factors were 13.8±0.7 mV (), 15.6±1 mV (), and 15.2±1 mV (), respectively.

Short-Term Modulation of I_Ks by PKC
In oocytes expressing I_Ks current, activation of PKC by 100 nmol/L PDD induced a biphasic effect on the currents. Figure 1A shows a typical experiment in which I_Ks (hminK) current response to PDD was assessed by pulsing repetitively to 30 mV every 30 seconds during PDD application. The I_Ks (hminK) current amplitude was transiently increased by 100 nmol/L PDD, reached a peak at about 20 minutes, and then declined to below control levels in the continued presence of 100 nmol/L PDD. This biphasic effect induced by 100 nmol/L PDD was seen in all hminK- or cminK-injected oocytes examined (n=16). The inset shows current traces from a single oocyte expressing I_Ks (hminK) current before, at the peak of the increase, and 1 hour after PDD application. During the potentiation phase the I_Ks (hminK) and I_Ks (cminK) current amplitudes reached a peak of 148.5±12.2% of control level (n=16) 20 minutes after the start of PDD perfusion. The I_Ks currents then declined to an amplitude of 52±7% (n=16) of control level after 1 hour of PDD application. The amplitude of I_Ks currents continued to decline in the presence of 100 nmol/L PDD (100 min). The capacitance of the oocytes was not significantly altered after 1 hour of PDD application (141±20 nF in control and 114±12 nF in PDD; n=10 each).

The enhancement of guinea pig I_Ks current by activators of PKC has been attributed to a negative shift in the voltage dependence of activation.¹⁴ We therefore examined the effect of PDD on I_Ks current activation. I_Ks tail current amplitudes were measured after test pulses to different potentials and the I-V relationship plotted before and after 100 nmol/L PDD application. I-V plots were constructed before, at the peak of I_Ks current potentiation, and 1 hour after PDD application. Figure 1B shows the average I-V relationships for 6 oocytes expressing the I_Ks (hminK) current. A fit of the averaged data with the Boltzmann equation showed that there were no significant differences in the V_1/2 and slope factor in the control, potentiated, or downregulated current response (Figure 1B legend). Similarly, I_Ks (cminK) current was modulated by 100 nmol/L PDD without changes in the voltage dependence of activation (n=6). Our data show that PDD has no effect on the voltage dependence of I_Ks (hminK) or I_Ks (cminK) current activation.

Modulation of I_Ks current by PDD was altered by both the duration of application and the dosage of PDD used. Figure 2A shows that a 5-minute application of 100 nmol/L PDD increased I_Ks (hminK) currents, to 173.5±3% of control level (n=4) with no evidence for current suppression. A second 5-minute PDD application increased I_Ks (hminK) current further: a 148±6% increase compared with the first PDD-induced peak and a 263±55% increase compared with the control current (n=3). Then 100 nmol/L PDD was applied continuously and I_Ks (hminK) current decreased to 61±6.6% of control current (n=3). It was also possible to separate the potentiation of I_Ks current from the suppression by use of low doses of PDD. Figure 2B shows a representative experiment in which I_Ks (cminK) current was upregulated by application of 5 nmol/L PDD for more than 100 minutes with no evidence for suppression. Similar effects were seen in 4 other oocytes, expressing either human or cat minK and exposed to 5 nmol/L PDD for a duration of 75 to 100 minutes. The average potentiation by 5 nmol/L PDD was 135±11% above control level when measured 75 minutes after PDD perfusion (n=5). The increase in I_Ks currents with 5 nmol/L PDD had a much slower time course than the transient increase seen with 100 nmol/L PDD (compare Figure 1A and 2A with Figure 2B). Brief 5- to 10-minute applications of 5 nmol/L PDD had no effect on I_Ks current (n=2).

View larger version (16K): [in this window] [in a new window]   Figure 2. PDD modulation of IKs current depends on time and concentration. A, IKs (hminK) current was stimulated by the standard voltage protocol, and the resulting current was measured at the end of each pulse. PDD (100 nmol/L) was applied for 5 minutes at the times indicated at the top of the figure. After washout of PDD, IKs (hminK) current continued to increase to a steady-state level. A second 5-minute PDD application had similar effects, and subsequent continuous PDD application decreased IKs (hminK) current to a level below control current. B, Application of 5 nmol/L PDD induced slow activation of IKs (cminK) current with no suppression.

Continuous application of 20 nmol/L PDD modulated I_Ks current in a biphasic manner similar to that seen with 100 nmol/L PDD, although the time course of the current potentiation effect was slightly slower (n=3, data not shown). These results show that the time and concentration of PDD application are important in determining its overall effects on I_Ks current.

In addition to PDD, 2 other PKC activators were used to examine the modulatory effects of PKC on I_Ks current. DOG and OAG are diacylglycerol analogues that activate PKC. Whereas a brief application (5 to 10 minutes) of 100 µmol/L DOG or OAG (n=5) had no effect on I_Ks currents, continuous perfusion of 100 µmol/L OAG and DOG induced a slow monophasic decrease in I_Ks current with no potentiation phase (Figure 3A). After 1 hour of OAG and DOG perfusion, the oocyte capacitance was not changed significantly (control: 117±15 nF, OAG- or DOG-treated: 97±9 nF; n=10 each). Compared with PDD, higher concentrations of DOG and OAG (50 to 100 µmol/L) were required to elicit the downregulatory effect, although the time course of the current decline was similar. Figure 3B summarizes the effects of different PKC activators on the amplitude of I_Ks currents. The extent of I_Ks current downregulation was comparable regardless of the PKC activator used.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of various PKC activators on IKs currents. A, Representative experiment showing that 100 µmol/L DOG decreased IKs (hminK) current without an initial potentiating phase. Inset shows current records at times indicated. Scale bars=500 nA and 2 seconds. B, Summary plot showing the effects of different PKC activators on IKs currents. Currents were measured at the end of a test pulse to 30 mV for 5 seconds at the times shown after application of PKC activators. These values were normalized to control currents before application of PKC activators. The inactive enantiomer of PDD (4-PDD) was included as a negative control. Number of experiments is shown above each bar.

PDD has been reported to have nonspecific, PKC-independent actions.²² However, perfusion of 100 nmol/L 4-PDD, an inactive enantiomer of PDD, had no effect on I_Ks current (Figure 3B). There was no effect on HERG current from 100 nmol/L PDD (data not shown). Preincubation of oocytes for 2 to 4 hours with 2 to 4 µmol/L of the PKC inhibitor staurosporine abolished the effects of 100 nmol/L PDD (n=3). The results suggest that modulation of I_Ks current by PDD is specific and requires PKC activation. Evidence will be presented later to confirm this with a specific PKC inhibitory peptide.

PKC and PKA Interact in Their Modulation of I_Ks
I_Ks current is modulated by other protein kinases such as PKA.²³ PKA increases guinea pig, rabbit, rat, and mouse I_Ks currents expressed in oocytes.¹⁸ I_Ks (hminK) and I_Ks (cminK) currents were also stimulated by PKA.¹⁵ Figure 4A shows the response of I_Ks (hminK) current to PKA activation, induced by 20 µmol/L forskolin and 500 µmol/L IBMX. Figure 4B shows the typical downregulatory effect on I_Ks (hminK) current after 1-hour exposure to 100 nmol/L PDD. Because I_Ks current is modulated by both PKC and PKA, we investigated the effects of PKC and PKA comodulation. After downregulation of I_Ks (hminK) current by 1-hour exposure to 100 nmol/L PDD, subsequent activation of PKA did not significantly increase I_Ks (hminK) current amplitude (Figure 4C). I_Ks current amplitudes were 49±5.5% of control after 1-hour of PDD application, and 54±6% of control in the presence of PDD and PKA activators (50 minutes after addition of forskolin plus IBMX; n=9). Likewise, in the continuous presence of forskolin and IBMX, 100 nmol/L PDD did not have any additional effect on I_Ks (hminK) currents even after 1-hour exposure (Figure 4D). PKA activation increased I_Ks current amplitudes to 273±57% of control (n=8), and subsequent PKC activation did not significantly alter the current magnitude (202±31% of control; n=4). The activation curves were not significantly altered by either treatment: with V_1/2=-6.9±4.9 mV for control, -6.0±2.3 mV for PKA activation, and -9.5±5.6 mV for both PKA and PKC modulation of I_Ks (cminK) current. When repetitive brief applications (5 minutes) of 100 nmol/L PDD (which induce I_Ks current potentiation) were followed by addition of PKA activators, no additional potentiation could be seen (162±9% of control after brief PDD application and 187±26% after subsequent PKA activation; n=4). The results show that the modulatory effects of PKC and PKA on I_Ks current are nonadditive and mutually exclusive.

View larger version (12K): [in this window] [in a new window]   Figure 4. Effects of PKC and PKA activation on IKs currents. A, PKA stimulates IKs (hminK) current. IKs (hminK) current was increased in the presence of 20 µmol/L forskolin plus 500 µmol/L IBMX. B, IKs (hminK) current modulation by PKC. Representative experiment shows that 1-hour application of PDD decreased IKs (hminK) current. C, Recordings from another oocyte shows that 1-hour application of PDD decreased IKs (cminK) current, and that subsequent application of PKA activators had little additional effect. D, Application of PKA activators increased IKs (hminK) current 2-fold, but subsequent application of PDD for 60 minutes has no additional effect. PDD indicates 100 nmol/L PDD; PKA, 20 µmol/L forskolin plus 500 µmol/L IBMX. Scale bars=500 nA and 2 seconds.

To study the comodulatory roles of PKA and PKC in more detail, specific protein kinase inhibitors were injected into oocytes expressing I_Ks (hminK) or I_Ks (cminK) currents. Injecting 120 to 250 µmol/L of the pseudosubstrate peptide inhibitors of PKA or PKC 8 to 12 hours before recording did not alter the expression level, I-V relationships, or the activation or deactivation kinetics of I_Ks currents (data not shown). Figure 5A shows that injection of a PKA-specific inhibitory peptide (PKAI) had no effect on PDD modulation of I_Ks (hminK) current, because 100 nmol/L PDD still elicited the typical biphasic response. In 4 PKAI-injected oocytes, I_Ks currents increased to 140±20% of control and then decreased to 73±27.5% of control after 1 hour of 100 nmol/L PDD. This is similar to results obtained in oocytes not injected with the inhibitory peptides. However, injection of a PKC-specific inhibitory peptide (PKCI) completely abolished both the up- and downregulatory effects of 100 nmol/L PDD (Figure 5B). In 6 PKCI-injected oocytes, I_Ks currents did not respond to 45- to 90-minute perfusion of 100 nmol/L PDD (105.5±3.2% of control 1 hour after PDD). The results confirm that PDD modulates I_Ks current selectively by activation of PKC and that the stimulatory effect of PDD is not caused by activation of PKA. Injection of PKCI did not affect the action of PKA on I_Ks currents, because forskolin plus IBMX still elicited a 161.5±13% increase of I_Ks current in the presence of PKCI (Figure 5C; n=5). Injection of PKAI did eliminate the stimulatory effect of forskolin and IBMX on I_Ks currents (91.5±28%, n=2), confirming the specificity of PKAI. These results suggest that PDD increases I_Ks current in a PKC-specific, but PKA-independent manner.

View larger version (12K): [in this window] [in a new window]   Figure 5. PDD modulation of IKs current is strictly PKC dependent. A, An oocyte injected with 250 µmol/L PKA inhibitory peptide (PKAI), PDD elicited an increase in IKs (hminK) current within 12 minutes. IKs (hminK) current then decreased to below control level after 46 minutes of PDD perfusion. B, Another oocyte injected with 250 µmol/L of a PKCI and perfused with 100 nmol/L PDD for 46 minutes produced no effect on IKs (cminK) current. C, In an oocyte injected with 250 µmol/L PKCI, PKA activators increased IKs (hminK) current 2-fold. PDD, 100 nmol/L PDD; PKA, 20 µmol/L forskolin plus 500 µmol/L IBMX. Scale bars=500 nA and 2 seconds.

Long-Term Downregulation of I_Ks by PKC
Continuous application of 100 nmol/L PDD decreased I_Ks (hminK) and I_Ks (cminK) currents with a very slow time course. A steady-state level of I_Ks current was not reached even after 2.5 hours application of PDD. We therefore examined the long-term effect of PDD on I_Ks current expression. Figure 6A and 6B show that overnight incubation (12 to 20 hours) of I_Ks (hminK)-expressing oocytes with 100 nmol/L PDD chronically reduced the I_Ks (hminK) current density. In 2 independent experiments, I_Ks (hminK) current densities were 45.8±11% and 37.7±4% of control currents after overnight treatment with 100 nmol/L PDD (n=25). The I-V relationships of I_Ks (hminK)-expressing oocytes treated overnight with 100 nmol/L PDD were not different from control hminK-injected oocytes, although oocyte capacitance was significantly decreased from 153±15 nF to 78±11 nF (n=15). Similar results were seen for oocytes expressing I_Ks (cminK). Overnight incubation with 100 µmol/L OAG also decreased I_Ks (hminK) current densities, although to a lesser extent (63±15% of control current; n=7). There was no effect on I_Ks (hminK) current density or oocyte capacitance after overnight incubation with either 5 nmol/L PDD (n=4) or 100 nmol/L 4-PDD (Figure 6A; n=3). After overnight incubation of 100 nmol/L PDD and extensive wash with control solution (>30 min), 20 µmol/L forskolin plus 500 µmol/L IBMX still increased the remaining I_Ks current amplitude by 153±9% (n=2), indicative of normally functioning I_Ks channels.

View larger version (30K): [in this window] [in a new window]   Figure 6. Long-term effects of PDD on IKs and HERG currents. PDD (100 nmol/L) was added to the oocyte incubation medium for 16 to 22 hours. A, Overnight, PDD incubation decreased IKs (hminK) current density by 45.8±11% of control oocytes (without PDD). Overnight incubation with 100 nmol/L 4-PDD, an inactive form of phorbol ester, had no significant effects on IKs (hminK) currents. Representative current traces from a control and a PDD-treated oocyte are shown on the right. Scale bars=1 µA and 2 seconds. B, Oocytes injected with hminK cRNA were treated with various PKC inhibitors 2 hours before overnight 100 nmol/L PDD incubation. Chele indicates 20 µmol/L chelerythrine; calp, 4 µmol/L calphostin C; and stau, 4 µmol/L staurosporine. C, Overnight PDD incubation (100 nmol/L) had no effect on the expression of HERG channels. Current traces showing control and PDD-treated oocytes are shown on the right. Tail current amplitudes were measured at -70 mV after a test pulse to 30 mV for 5 seconds. Scale bars=1 µA and 2 seconds.

We further addressed the specificity of the PDD long-term effect by using several membrane-permeable PKC inhibitors. Oocytes were incubated with different PKC inhibitors 2 to 3 hours before overnight PDD incubation. Figure 6B shows that nonspecific kinase inhibitors such as staurosporine, as well as more selective PKC inhibitors such as calphostin C and chelerythrine, antagonized the downregulation of I_Ks current by overnight treatment of 100 nmol/L PDD. The oocyte capacitance was not altered significantly by overnight exposure to 100 nmol/L PDD in the presence of PKC inhibitors (n=18). Incubation of hminK-injected oocytes with chelerythrine alone had no effect on the current amplitude nor the voltage dependence of activation (n=11). To explore the PKC downregulation on a different K⁺ channel, we treated oocytes expressing HERG with 100 nmol/L PDD for 18 to 22 hours. The HERG current density, kinetics, and the oocyte capacitance, were similar for the PDD-treated and control HERG-expressing oocytes (Figure 6C). Our results suggest that long-term PKC activation specifically and irreversibly attenuates the I_Ks current.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first report of an increase in I_Ks current by PKC modulation in a species with a serine at position 102; however, the modulation of I_Ks (hminK) and I_Ks (cminK) currents has not been examined to date. Other species may have a similar pattern of modulation by PKC. We observe no change in the voltage-dependence of activation of I_Ks (hminK) and I_Ks (cminK) after modulation by PKC, in contrast with the negative 6 to 10 mV shift seen in guinea pig minK, I_Ks current.¹⁴ Our results are similar to the results in guinea pig myocytes in which an increase in I_Ks current by phorbol ester was observed in the absence of any voltage shift in the I-V curve. However that study did report a change in the slope of the activation curve that we did not observe.²⁴ These differences presumably reflect the different KvLQT1 and minK isoforms found in these preparations. Our results suggest that phosphorylation at serine 102 alone is not sufficient to account for the complex, biphasic response of I_Ks current to PKC modulation. There are 4 consensus PKC sites in the partial Xenopus KvLQT1 sequence² and 7 consensus PKC sites in the full-length human KvLQT1.^{1 2 25} It is therefore possible that these additional phosphorylation sites are involved in the biphasic PKC effect. Mutagenesis studies are required to address this possibility.

PKC Modulation of I_Ks Appears to Involve Multiple Phosphorylation Sites
A surprising finding was that OAG and DOG, which also activate PKC, only produce a downregulation of the I_Ks current. It has been reported that in rat cardiac myocytes, OAG and phorbol esters have different potency and mechanisms of action on the L-type Ca²⁺ channels. This was postulated to reflect different forms of PKC being activated by the different PKC activators.²⁶ Multiple forms of Ca²⁺-dependent and independent PKC enzymes are found in oocytes²⁷ and in cardiac myocytes.^{28 29} It is possible that specific PKCs may have different sensitivities to activation by various PKC activators,^{30 31} and that once activated, different PKCs may phosphorylate different sites. According to this hypothesis, PDD could activate both high-and low-sensitivity PKC isoforms (sensitivity relative to PDD), and OAG/DOG would only be able to activate the low-sensitivity PKC isoform. The high-sensitivity PKC isoform may phosphorylate a unique site on minK-KvLQT1 that would produce potentiation of current. On the other hand, the low-sensitivity PKC isoform may phosphorylate a site common to PDD and OAG/DOG activation that would downregulate the current. This model would explain why low concentrations of PDD upregulates the current, by activating the high-sensitivity PKC isoform and phosphorylating the unique potentiating site. The low-sensitivity isoform of PKC would require higher concentrations of PDD and longer exposure for its activation. This is supported by the data showing that brief repeated applications of 100 nmol/L PDD only elicit potentiation of I_Ks current, but brief applications of 100 µmol/L OAG and DOG have no effect. Biphasic regulation of a different K⁺ channel by PKC has been reported previously. PKC activation leads to an upregulation followed by a downregulation of the Aplysia Kv1.1a channel, similar to our data on I_Ks current. It was found that 2 different phosphorylation sites are involved in the modulation of Kv1.1a.³²

Mechanisms for Long-Term Downregulation of I_Ks by PKC
There was a specific decrease in oocyte capacitance in oocytes whose I_Ks current has been downregulated by prolonged treatment with 100 nmol/L PDD, suggesting that the decrease in I_Ks current expression may be related to the internalization of surface membrane. In support of this, overnight incubation with 5 nmol/L PDD, or 100 nmol/L 4PDD, or inclusion of PKC inhibitors with 100 nmol/L PDD, all of which do not reduce I_Ks current, also produce no decrease in oocyte capacitance. If PKC phosphorylation of minK-KvLQT1 channels leads to internalization of the channels, perhaps the mechanism is similar to the phosphorylation-induced desensitization of the ß-adrenergic receptors. ß-Adrenergic receptors can be phosphorylated at multiple sites by several kinases such as ßARK and PKA.³³ The phosphorylated receptor first exhibits a reduction in the agonist affinity, and then the receptors are internalized in the continued presence of agonist. However, it should be noted that in the present study, although there is a 49% (n=15) decrease in oocyte capacitance; most of the reduction in I_Ks current is not occurring through this mechanism, because current density levels (normalizing for this reduction in membrane area), as shown on Figure 6, still decrease dramatically with long-term treatment with PDD. Other possible mechanisms to explain this reduction in current density include a turnover of I_Ks channels through synthesis and internalization modulated by PKC stimulation so that there is a large reduction in channel synthesis in addition to enhanced internalization or degradation. Whatever the precise mechanism, it appears to be specific for I_Ks, because oocytes expressing HERG and treated overnight with PDD show no effects on the I_Kr current or the oocyte capacitance.

Mutually Exclusive Modulation of I_Ks by PKC and PKA
An interesting finding was the lack of comodulation by PKC and PKA on I_Ks current. We observed potentiation of both I_Ks (hminK) and I_Ks (cminK) currents by activators of PKA.¹⁵ Because there is no consensus PKA phosphorylation site on minK, it is likely that the PKA phosphorylation site(s) is on KvLQT1. It has been shown recently that KvLQT1, expressed alone or with minK, is stimulated by PKA.²⁵ Our results show that after PKC modulation of I_Ks current, the current is no longer sensitive to PKA. The opposite is also true, because after I_Ks current potentiation by PKA, PKC is unable to modulate the current. Convergent modulation of ion channels by different protein kinases have been reported. For example, the type II Na⁺ channel can be modulated by PKA after it is first phosphorylated at a PKC site.³⁴ Nevertheless, the mutually exclusive actions of PKA and PKC on I_Ks current appears to be a novel mechanism of ion channel regulation. This exclusivity ensures that I_Ks current is modulated by either PKA or PKC, but not by both at the same time. Moreover, the order of PKA or PKC modulation is pivotal in determining the effect seen. In the partial sequence of Xenopus KvLQT1, a putative PKA site (serine 390) is flanked by 2 putative PKC sites (threonine 381 and serine 399).² Because the PKA and PKC sites are located close to one another, steric hindrance of the negatively charged phosphate groups may prevent all sites from being phosphorylated. This would be one way to explain the mutually exclusive effects of PKC and PKA activation on I_Ks current. Alternatively, a conformational change caused by phosphorylation by a kinase may hide or block other sites from additional phosphorylation.

PKA activation by the ß-adrenergic receptors stimulates I_Ks in cardiac myocytes of various species.^{4 16} It has also been shown that PDD activates I_Ks in guinea pig myocytes,^{22 25} whereas PDD decreases I_Ks in mouse myocytes.¹³ It is less clear how I_Ks is regulated in higher mammals, such as the cat and the human. Our results imply that regulation of human and cat I_Ks by PKC may be different from other species. It appears that although the serine/asparagine 102 site is probably involved, the exact molecular mechanism of PKC modulation of I_Ks currents is likely to be more complicated.

In summary, we have investigated the modulation of human and cat minK I_Ks currents by phosphorylation. PKC modulation of I_Ks currents is complex and depends on the time and concentration of PDD. Our results suggest that there are at least 2 functionally distinct PKC sites on KvLQT1-minK. One that potentiates I_Ks current, and another that downregulates it. Long-term exposure to PKC activators leads to irreversible downregulation of I_Ks current. PKA increases I_Ks current amplitude, and the effects of PKA and PKC are mutually exclusive. This suggests that PKA and PKC may phosphorylate common sites, or that phosphorylation of one site occludes phosphorylation of other sites. Given the importance of I_Ks in cardiac action potential repolarization, and its role in long-QT syndrome, understanding the regulation of human I_Ks may shed light on its physiological and pathophysiological roles. The ability to up- or downregulate I_Ks could be a means of controlling the duration of action potential in human heart. Further studies on the regulation of minK-KvLQT1 from different species coexpressed in mammalian cells should provide a better understanding of the modulation of this important cardiac current.

	Acknowledgments

 
We thank Huai-Ping Ling for providing human and cat minK cRNA and Dr Thomas Colatsky for encouragement and support.

Received January 27, 1998; accepted August 3, 1998.

	References

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