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. 1999 Oct 15;520(Pt 2):439–449. doi: 10.1111/j.1469-7793.1999.00439.x

Protein kinase C regulation of K+ currents in rat ventricular myocytes and its modification by hormonal status

Y Shimoni 1
PMCID: PMC2269583  PMID: 10523413

Abstract

  1. The effects of protein kinase C (PKC) activation on cardiac K+ currents were studied in rat ventricular myocytes, using whole-cell voltage clamp methods. Control rats were compared to hypothyroid or diabetic rats, in which PKC expression and activity were enhanced.

  2. In control myocytes, two calcium-independent outward K+ currents, the transient It and the sustained Iss, were attenuated by 18.9 ± 2.0 and 16.8 ± 3.5%, respectively (mean ±s.e.m.), following addition of a synthetic analogue of diacylglycerol, DiC8 (20 μm). In myocytes from hypothyroid or diabetic rats, It and Iss were not affected by DiC8.

  3. The effects of DiC8 were restored in myocytes from thyroidectomized rats by injection of physiological doses of tri-iodothyronine (T3; 10 μg kg−1 for 6–8 days). Incubating cells from diabetic rats with 100 nm insulin for 5–9 h also restored the ability of DiC8 to attenuate It and Iss.

  4. The attenuation of K+ currents by DiC8 in control cells was absent in the presence of a peptide known to inhibit the translocation of the isoform PKCɛ (EAVSKPLT, 24 μm, introduced through the recording pipette). A scrambled peptide (LSETKPAV) was without effect.

  5. Under hypothyroid conditions the inhibitory peptide restored the effects of DiC8 on It and Iss. These currents were attenuated by 11.9 ± 1.5 and 9.8 ± 1.5%, respectively, which was significantly (P < 0.001) more than without the peptide or with the scrambled peptide.

  6. These results show that the PKC-mediated suppression of cardiac K+ currents is normally mediated by PKC ɛ translocation. This effect is absent under hypothyroid and diabetic conditions, presumably due to prior PKC activation and translocation. A PKCɛ translocation inhibitor restores the ability of DiC8 to attenuate K+ currents under hypothyroid conditions. This presumably reflects a (partial) reversal of a chronic translocation and a shift in the balance between PKC and its anchoring proteins.

Protein kinases have long been known to directly modulate the function of various cardiac ion channels (Walsh & Kass, 1988; Shearman et al. 1989; Bourinet et al. 1992; Qu et al. 1994; Murray et al. 1994; Lo & Numann, 1998; Middleton & Harvey, 1998). In rat ventricle, the two major calcium-independent repolarizing currents, the transient outward current It and the sustained, quasi-steady-state current Iss (Apkon & Nerbonne, 1991) are attenuated when PKC is activated by phorbol esters (Apkon & Nerbonne, 1988; Nakamura et al. 1997). These effects are presumably due to direct channel phosphorylation by the activated kinase (Nakamura et al. 1997), based on the fact that many kinase consensus sites have been identified in the course of cloning various potassium channels (Kemp & Pearson, 1990; Lo & Numann, 1998).

Protein kinase C has at least 11 known isoforms (Harrington & Ware, 1995; Mellor & Parker, 1998), with several of these known to be expressed in rat heart (Disatnik et al. 1994; Steinberg et al. 1995). It is known that the activation of PKC involves its translocation between different subcellular compartments, usually from the cytosol to cellular membranes (Mochly-Rosen & Gordon, 1998). This translocation occurs between isoform-specific anchoring proteins. The inactive PKC is anchored to proteins labelled RICKS (receptors for inactive C-kinases). Activation results in translocation and binding to other anchoring proteins labelled RACKS (receptors for activated C-kinases; Mochly-Rosen & Gordon, 1998). Moreover, isoform-specific inhibitory peptides have been designed and used to specifically inhibit the translocation of several PKC isoforms, and thus to block PKC actions in different cells (Johnson et al. 1996; Yedovitsky et al. 1997).

In recent years there has been mounting evidence for changes which occur in the expression and activity (associated with subcellular redistribution) of several PKC isoforms in rat ventricular cells, either during post-natal development (Rybin & Steinberg, 1994) or under different pathological conditions. These include heart failure, ischaemia/hypoxia and hypertrophy (Gu & Bishop, 1994; Rouet-Benzineh et al. 1996; Yoshida et al. 1996; Goldberg et al. 1997). Imbalances in hormonal levels have also been found to affect PKC. Thus, under hypothyroid (Rybin & Steinberg, 1996) or diabetic (Malhotra et al. 1997) conditions there is an upregulation of the expression and activity of the ε isoform of PKC (PKCε). This is the major isoform in the adult rat ventricle (Bogoyevitch et al. 1993), and its activation has been shown to affect cardiac function (Puceat et al. 1994; Johnson & Mochly-Rosen, 1995). The upregulation of PKCε presumably entails a chronic translocation to cellular membranes, although this has not been directly shown. It has been suggested that once translocated, PKC is protected from degradation by proteases (Blobe et al. 1996), thus enabling a sustained translocation.

Since many pathological changes or adaptations in cardiac function have been shown to involve alterations in PKC activity or expression, it is of great interest and importance to investigate whether the modulation of K+ currents by PKC is altered under some of these pathological conditions.

The present study was designed primarily to identify whether the direct effects of PKC activation on It and Iss in rat ventricle are altered in hypothyroid and diabetic conditions. In addition, a major aim was to establish whether translocation of PKCε is involved in attenuation of either of these currents, and whether changes in PKC under hypothyroid conditions can be associated with changes in the translocation process. The first aim was addressed in experiments comparing the effects of PKC activation (by a synthetic analogue of diacylglycerol, the endogenous activator of PKC) in cells from control rats and from rats made hypothyroid or diabetic. The second aim was addressed in experiments using a peptide which specifically inhibits the translocation of PKCε (Johnson et al. 1996). The results show that a deficiency in either thyroid hormone or insulin is associated with an attenuation or abolition of the direct effects of PKC activation on It and Iss The results also indicate that the translocation of PKCε mediates the attenuation of both It and Iss by PKC activators. Finally, under hypothyroid conditions the peptide inhibitor of PKCε translocation can restore the attenuating effects of a PKC activator on the two K+ currents, presumably by partially reversing a chronic translocation of PKCε.

METHODS

All experiments were carried out according to the guidelines laid down by the Animal Care Committee of the University of Calgary.

Experimental groups

The experiments were done using single cardiac myocytes obtained from male Sprague-Dawley rats (200-250 g), divided into three groups: control, untreated rats; rats which had been thyroidectomized 4-6 weeks prior to the experiments; rats which had been made diabetic (insulin-deficient) by a single i.v. injection of 100 mg kg−1 streptozotocin (STZ), given 7-12 days prior to experiments. A subgroup of thyroidectomized rats was injected i.p. with tri-iodothyronine (T3; 10 μg kg−1) for 6-8 days prior to experiments, to normalize their T3 levels. Thyroidectomy was performed after the rats were anaesthetized with a mixture (given by i.p. injection) of ketamine (90 mg kg−1) and xylazine (10 mg kg−1). The animals were kept in a warm room (23-27°C) and given 5 % CaCl2 in their drinking water for 48 h post-operative. The thyroid or diabetic status of thyroidectomized or STZ-injected rats was confirmed by collecting blood on the day of experiment following removal of the heart. Plasma T3 levels were measured using standard radioimmunoassay (Foothills Hospital Clinical Laboratory). In 13 thyroidectomized rats, T3 levels were 0.42 ± 0.04 nm (mean ±s.e.m.), whereas in four such rats injected with T3, the plasma levels were significantly (P < 0.005) elevated, with a value of 1.90 ± 0.9 nm, which is in the normal range. The diabetic status of rats injected with STZ was confirmed by measurements of plasma glucose and insulin, which were elevated and reduced, respectively, in comparison to normal values (see Shimoni et al. 1998). All animals were monitored, with normal food and water intake indicating their health.

Cell isolation

Rats were anaesthetized by methoxyflurane inhalation and killed by cervical dislocation. Hearts were removed and the aortas cannulated on a Langendorff apparatus for retrograde coronary perfusion (at 37°C, 70 cmH2O pressure). The hearts were perfused for 5 min with a solution containing (mm): 120 NaCl; 5.4 KCl; 2.8 sodium acetate; 1 MgCl2; 1 CaCl2; 5 Na2HPO4; 24 NaHCO3; 5 glucose. This was followed by 10 min perfusion in the same solution from which CaCl2 was omitted, and then for 6-8 min in the same solution to which was added the digestive enzymes collagenase (10 U ml−1, Yakult Honsha, Tokyo) and protease (0.01 mg ml−1, Sigma type XIV), as well as 20 mm taurine and 40 μm CaCl2. The free wall of the right ventricles was then removed and cut into smaller pieces for further digestion in a shaker bath (at 37°C), in the same calcium-free solution as before, with the addition of 0.1 mm CaCl2, 50 U ml−1 collagenase, 0.1 mg ml−1 protease, 20 mm taurine and 10 mg ml−1 albumin. Aliquots of cells were removed over the next 30-50 min, and stored in the same solution, with no enzymes, containing 0.1 mm CaCl2, 20 mm taurine, 10 mg ml−1 albumin.

Current recording

Cells were placed in a bath on the stage of an inverted microscope and perfused with a solution containing (mm): 150 NaCl; 5.4 KCl; 1 CaCl2; 1 MgCl2; 5 Hepes, 5.5 glucose, titrated with NaOH to a pH of 7.4. CdCl2 (0.3 mm) was added to block the L-type calcium current. Recordings were made with an LM-EPC7 amplifier, using the whole-cell suction electrode in voltage clamp mode. Pipettes (2-4 MΩ resistance) were filled with solutions containing (mm): 110 potassium aspartate; 30 KCl; 4 Na2ATP; 1 MgCl2; 5 Hepes, 10 EGTA; 1 CaCl2, titrated with KOH to a pH of 7.2. In some experiments the perforated-patch method (Horn & Marty, 1988) was used. In these experiments, stock solutions were made by dissolving 5 mg nystatin in 50 μl DMSO. This was then diluted in the same pipette solution as above (3 μl in 1 ml). Since under these conditions EGTA is ineffective (being too large to pass through the pores formed by nystatin), the membrane-permeant calcium chelator BAPTA AM (5 μm) was used to buffer intracellular calcium. All recordings were done at 21-22°C. In the experiments with peptides in the filling solution, the conventional suction method had to be used.

In the present experiments two currents were measured: the calcium-independent transient outward current It, measured as the peak outward current in response to depolarizing steps from -80 to +50 mV. CdCl2 in the perfusate and EGTA in the pipette blocked any calcium-dependent outward currents. The second current was the calcium-independent delayed rectifier (sustained) current Iss, measured as the current magnitude at the end of a 500 ms pulse to +50 mV. Series resistance was minimized by using low resistance electrodes, and by electronic compensation. The present set of experiments involved recordings for up to 40 min. In the whole-cell mode of recording, series resistance (Rs) may increase over time. A convenient way of monitoring Rs changes was to periodically interpose pulses to -110 mV, which elicit a third K+ current, IK1. Any increase in Rs results in a reduction in IK1. Since IK1 was not affected by any of the protocols used, changes in Rs could be monitored directly as changes in IK1 (see below). Results were discarded when changes of more than 10 % occurred during the experimental protocols. Interposing negative pulses led to a slight ‘rebound’ enhancement of the subsequent outward currents. This accounts for the uneven current magnitudes when plotted vs. time, as illustrated in the following figures. All currents were digitized (at 2 kHz) and stored on a PC for subsequent analysis.

Cell capacitance was measured by recording currents elicited by 5 mV steps (from -80 mV), digitized at 10 kHz. Capacitance was calculated using electronic integration of the currents. Capacitance was used to correct for cell size; dividing current magnitudes by cell capacitance gave current densities.

Drugs and reagents

PKC activation was obtained with 1,2-dioctanoyl-rac-glycerol (C8:0, termed here DiC8), a synthetic membrane-permeant analogue of diacylglycerol, the endogenous activator. DiC8 and insulin were obtained from Sigma. Tri-iodothyronine (T3), chelerythrine, bisindolylmaleimide I and the peptide inhibitor of PKCε translocation (EAVSKPLT) were obtained from Calbiochem. The scrambled peptide used as a control (LSETKPAV) was a generous gift from Dr M. Walsh (University of Calgary). All drugs were prepared as concentrated stocks, in either water or DMSO, and frozen until the final dilution just before use. Final concentrations of DMSO did not exceed 0.03 %, which had no effects on its own.

Statistics

Results were summarized as means ±s.e.m., and the significance of any differences was evaluated using a Student's unpaired t test (P < 0.05 considered significant).

RESULTS

Effects of PKC activation on It and Iss

Previous studies (Apkon & Nerbonne, 1988; Nakamura et al. 1997) showed that activation of PKC by phorbol esters attenuates It and Iss in rat ventricular myocytes. In the present experiments the activation of PKC was studied using DiC8, a membrane-permeant synthetic analogue of diacylglycerol, which is the endogenous activator. The advantage of this is that the effects can be reversed upon wash-out, in contrast to the irreversible effects of phorbol esters. In the present study, DiC8 was found to attenuate both It and Iss. However, the effects on the two currents seemed to be independent, since in some cells both currents were attenuated, whereas in others mainly It was affected. There were also apparently different rates of wash-out (see below). In 14 cells the addition of 20 μm DiC8 attenuated It by 18.9 ± 2.0 %, and Iss by 16.8 ± 3.5 %. An example is shown in Fig. 1.

Figure 1. The effects of the PKC activator dioctanoyl-rac-glycerol (DiC8) on transient and sustained outward currents in control myocytes.

Figure 1

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A, superimposed current traces in response to pulses (at 0.2 Hz) from -80 to +50 mV given before and 7 min after addition of 20 μm DiC8 (denoted by *). This recording was made using conventional, ruptured-patch whole-cell recording. The arrow to the left of the traces denotes zero current level (here and in following figures). B, same protocol as in A, in a different cell, using the nystatin perforated-patch recording method. Outward currents are upward deflections, with * denoting the trace obtained following DiC8. Also shown is a trace following wash-out of DiC8 (w/o). Note that peak current, It, is fully recovered at a time when the steady-state current is still only partially recovered, suggesting independent modulation of the two currents. This panel also shows responses to a pulse to -110 mV, which elicits a third K+ current, IK1, seen as the downward (negative) current traces. This current is unaltered by DiC8. C, the time course of changes in magnitude of It (^) and Iss (•) (same cell as B), showing the reversible attenuation elicited by exposure to 20 μm DiC8.

Figure 1A shows recordings from a cell in which mainly the peak current was attenuated. In the whole-cell (ruptured patch) method of recording, some essential cofactors required for PKC activation may be dialysed, since the pipette volume is much larger than the cell volume. These experiments were therefore repeated using the nystatin perforated-patch method of recording (Horn & Marty, 1988), in which no dialysis can occur. In four cells, 20 μm DiC8 attenuated It by 23.8 ± 4.8 %, and Iss by 23.4 ± 7.0 %. The slight enhancement of the attenuation of both currents using the nystatin (perforated-patch) method (in comparison to the whole-cell method) was not significant (P > 0.05). Sample current traces obtained with the nystatin method, before and after addition of DiC8, as well as following wash-out, are shown in Fig. 1B. In this cell both currents were substantially attenuated (denoted by *). However, upon wash-out (w/o) the peak current recovered before full recovery of Iss, indicating the independent effects of PKC on the two currents. Figure 1B also shows superimposed (downward) current traces, obtained in response to hyperpolarizing pulses to -110 mV, indicating that no change occurs in IK1 (and in Rs). Figure 1C shows the time course of the effect (same cell as in B), as well as the reversal following wash-out. In both types of recording, EGTA and BAPTA rule out the involvement of any PKC isoforms which are calcium dependent.

It was important to establish that DiC8 affects the measured K+ currents by activation of PKC, since possible direct actions on ion channels have been reported (Bowlby & Levitan, 1995). This was done in a series of experiments in which DiC8 was added after pre-exposure of the cells to PKC inhibitors. Two different inhibitors were used. In nine cells pre-exposed to 2 μm chelerythrine the effects of DiC8 were essentially abolished. The mean reduction of It by 20 μm DiC8 was by only 3.4 ± 0.9 %, with no reduction in Iss (< 1 %). In eight cells pre-exposed to 100 nm bisindolylmaleimide the results were the same. DiC8 produced a mean reduction of It of only 2.8 ± 1.1 % with no attenuation of Iss. An example from one of these cells is shown in Fig. 2.

Figure 2. The effects of DiC8 are absent in the presence of a PKC inhibitor.

Figure 2

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Recordings are shown from a cell pre-incubated with 100 nm bisindolylmaleimide I for 30 min. A, superimposed current traces in response to steps to +50 mV (upward deflections) and to -110 mV before and 15 min after addition of 20 μm DiC8 show no attenuation in any current. B, the magnitudes of It, Iss and IK1 are shown as a function of time, following addition of DiC8, in the presence of the PKC inhibitor bisindolylmaleimide. No change is seen in any of these currents, in contrast to the effects in Fig. 1, indicating that DiC8 acts by activation of PKC.

Figure 2A shows superimposed current traces before and 18 min after addition of 20 μm DiC8. Figure 2B shows the current magnitudes as a function of time, for the three potassium currents measured, as indicated. The blocking of the action of DiC8 by chelerythrine and bisindolylmaleimide indicates that in these cells under these conditions, the effects of DiC8 (as in Fig. 1) are obtained primarily by activation of PKC. Further evidence for this is presented below.

PKC activation in cells from hypothyroid rats

The major isoform of PKC in rat ventricular cells is the ε subtype (Bogoyevitch et al. 1993). Thyroid hormone has been shown to repress this isoform (Rybin & Steinberg, 1996), so that, under conditions of reduced hormone levels, both the expression and activity of PKCε are increased. Based on these findings, the effects of DiC8 on It and Iss were examined in myocytes from hypothyroid rats. We had earlier reported (Shimoni & Severson, 1995) that It and Iss are attenuated under hypothyroid conditions. The present experiments examined the regulation of these currents by PKC activation under hypothyroid conditions. In 17 cells (ruptured-cell method), the addition of 20 μm DiC8 produced very little or no attenuation of either It or Iss, in marked contrast to the effects in control cells. The mean reduction of It was by only 0.7 ± 0.4 %, and of Iss by 0.6 ± 0.4 %. Several additional recordings were made using the nystatin perforated-patch method. Even with this method, there was very little attenuation of either current. In four cells, the mean reduction of It was by 2.3 ± 2.3 %, and Iss was attenuated by 3.5 ± 3.5 % (not significantly different from that in cells from hypothyroid rats recorded in the whole-cell ruptured-patch recording method). Figure 3 shows an example of this result. Figure 3A shows superimposed traces in response to pulses to +50 mV, before and after application of 20 μm DiC8. The downward traces show that IK1 was unchanged, indicating no change in Rs. Figure 3B shows the time course of current magnitude following DiC8 application, indicating the lack of change in either It or in Iss.

Figure 3. The effects of DiC8 in a myocyte from a hypothyroid rat.

Figure 3

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A, superimposed current traces in response to pulses to +50 mV (upward traces) and to -110 mV (downward traces), before and 7 min after addition of 20 μm DiC8 to the bath. B, current magnitudes as a function of time, after addition of DiC8: ^, It; •, Iss. Note the lack of effect, as compared to the attenuation shown in Fig. 1.

In additional experiments, we verified that the results in thyroidectomized rats are indeed a result of the deficiency in T3. Thyroidectomized rats were given T3, by six to eight daily subcutaneous injections (10 μg kg−1), which increased their plasma T3 levels to normal (see Methods). In cardiomyocytes obtained from these rats, it was found that T3 injection significantly (P < 0.001) increased both It and Iss. The respective values for these currents (at +50 mV) were 18.1 ± 1.2 and 6.8 ± 0.3 pA pF−1 in cells (n= 29) from thyroidectomized rats, whereas in cells (n= 34) from thyroidectomized rats receiving T3 replacement the values were 24.7 ± 1.5 and 9.4 ± 0.5 pA pF−1. In addition to increasing current densities, T3 treatment was found to restore the sensitivity of these currents to DiC8. In 11 cells, 20 μm DiC8 attenuated It by 14.6 ± 1.5 %, and Iss was reduced by 23.7 ± 2.5 %. This is not significantly different than in control rats, and indicates that it is the T3 deficiency following thyroidectomy that abolishes the effects of PKC activation on It and Iss. This result is shown in Fig. 4, which also shows that repeated DiC8 exposures, separated by wash-out, are equally effective in attenuating the two currents.

Figure 4. The effects of DiC8 in a myocyte from a thyroidectomized rat with T3 replacement.

Figure 4

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A, time course of changes in It (^), Iss (•, positive values) and IK1 (•, negative values). Note that the attenuation of It and Iss by DiC8 is restored following T3 replacement in thyroidectomized rats. This figure also shows that the effect of DiC8 is reversible and can be repeated following a second exposure. B, superimposed current traces in response to pulses from -80 to +50 mV and to -110 mV, before, during and after wash-out of 20 μm DiC8 (corresponding to points a, b and c in A). C, summary histogram showing the attenuation (%) of It (left) and Iss (right) by DiC8 in control cells (open columns), in cells from thyroidectomized rats (hatched columns) and in cells from thyroidectomized rats after replacement of T3 (cross-hatched columns).

PKC activation in cells from STZ-treated diabetic rats

Following these results, it was of interest to examine whether PKC upregulation in other pathological conditions has the same effects in terms of modulating It and Iss. It has long been known that PKC activity is altered in diabetes (Xiang & MacNeill, 1992). Initially, the βII isoform of PKC was reported to be increased in diabetes (Inoguchi et al. 1992). More recently, an increased expression and activity of the ε isoform has also been found in diabetic conditions (Malhotra et al. 1997). In the present experiments, nine cells from diabetic rats were exposed to 20 μm DiC8. As in the hypothyroid conditions, adding the PKC activator to cells from diabetic rats produced a much smaller (P < 0.05) attenuation of both currents, compared to the effects in cells from normal rats. The mean value of It attenuation was 5.1 ± 2.0 %, and Iss was reduced by 5.0 ± 2.0 %. An example is shown in Fig. 5. Figure 5A shows superimposed current traces (also showing no change in IK1), whereas Fig. 5B shows the current magnitudes as a function of time.

Figure 5. The effects of DiC8 in a myocyte from a STZ-treated diabetic rat.

Figure 5

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A, superimposed current traces obtained in response to pulses from -80 to +50 mV before and 7 min after addition of 20 μm DiC8. Also shown are the responses to a pulse to -110 mV (downward current traces). As in the hypothyroid myocytes, there is no attenuation in It and Iss. B, current magnitudes as a function of time (^, It; •, Iss) during and following addition of DiC8 to the bath.

In earlier work (Shimoni et al. 1998), we found that incubation of ventricular cells from diabetic rats with insulin can restore both It and Iss, after a period of 5-6 h or longer. In the present experiments we examined whether the restored currents also regain their sensitivity to PKC activation. Cells from diabetic rats were incubated for 6-9 h with 100 nm insulin. When 20 μm DiC8 was added after exposure to insulin, it was found that both currents were attenuated. In five cells, It was attenuated by 15.2 ± 4.1 %, and Iss was attenuated by 22.3 ± 5.8 %, which is significantly more (P < 0.05, for both currents) than the effects of DiC8 before insulin. An example and the summary data are shown in Fig. 6.

Figure 6. The effects of DiC8 in a cell from a diabetic rat following incubation for 6 h with 100 nm insulin.

Figure 6

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A, time course of It and Iss before and following addition of 20 μm DiC8. The currents are attenuated, and the effect is reversible upon wash-out. B, superimposed current traces (in response to steps to +50 and -110 mV), from times corresponding to points a and b in A. C, summary histogram showing the attenuation (%) of It (left) and Iss (right) in control cells (open bars), in cells from STZ-treated diabetic rats (hatched columns), and in the latter following 5-9 h incubation with 100 nm insulin (cross-hatched columns). Insulin restores the attenuation of the currents by DiC8, which is absent in untreated diabetic cells.

The results so far indicate that the effects of PKC activation on It and Iss are altered under hypothyroid and diabetic conditions, in which PKC activity has been shown to be enhanced. In particular, the activity of the ε isoform has been shown to be enhanced in both conditions (Rybin & Steinberg, 1996; Malhotra et al. 1997). This raises several questions: can the involvement of PKCε in attenuating these currents be demonstrated directly? Does the enhanced activity of PKCε under hypothyroid conditions entail a chronic translocation to its RACK? If so, this may prevent further translocation following addition of DiC8 and thus block the effects of DiC8 on the two K+ currents in hypothyroid and diabetic conditions. If there is a chronic translocation of PKCε, it may be possible to reverse this by (partly) inhibiting the interaction between PKC and its RACK. This may restore the ability of PKC activation to attenuate the two K+ currents (see Discussion). These questions were addressed by using the recently designed peptide, EAVSKPLT, which is targeted against the binding of PKCε to its RACK (Johnson et al. 1996).

PKC activation in the presence of the translocation inhibitor

Control cells

Experiments were done in which the specific PKCε inhibitory peptide EAVSKPLT was included in the recording pipette. The concentration used was 24 μm, which is the effective intracellular concentration reported by Johnson et al. (1996). In the present experiments, the effects depended on adequate diffusion from the pipette into the cell interior, so that the actual final intracellular concentration is unknown. However, in these experiments the inclusion of the inhibitor of PKCε translocation in the pipette effectively blocked the attenuation of both It and Iss by DiC8. Figure 7 illustrates this result.

Figure 7. Inhibition of the effect of DiC8 by a peptide which blocks PKCε translocation.

Figure 7

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A, current traces recorded from a control myocyte in response to pulses (from -80 mV) to +50 mV (eliciting It and Iss), and to -110 mV (eliciting IK1). The pipette solution contained 24 μm of the peptide EAVSKPLT. The superimposed traces were obtained before and 6 min after addition of 20 μm DiC8 to the bath, and indicate that DiC8 is without effect in the presence of the translocation inhibitory peptide. B, time course of It and Iss magnitudes following addition of DiC8, with EAVSKPLT in the pipette.

In four cells in which recordings were made with the inhibitory peptide, there was no attenuation of It or Iss at all, following addition of DiC8, whereas in eight other cells there was a very small residual attenuation, possibly due to limited diffusion from the pipette. In 12 cells, the mean attenuation of It was by 3.0 ± 0.9 %, whereas Iss was attenuated by 2.3 ± 1.1 %. These values are significantly (P < 0.001) smaller than the attenuation obtained when DiC8 is added under normal recording conditions. In control experiments, a scrambled peptide of the same composition and size (LSETKPAV) was included in the pipette filling solution. Under these conditions the addition of DiC8 was again found to reversibly attenuate It and Iss (n= 4), although in this group Iss was less affected by DiC8. An example is shown in Fig. 8.

Figure 8. Attenuation of It by DiC8 maintained in a control cell in the presence of a scrambled peptide.

Figure 8

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In this cell there was very little effect on Iss. A, the time course of changes in current magnitude shows a reversible attenuation of It by DiC8 in the presence of the scrambled peptide. B, superimposed current traces (same cell as A) are shown (same voltage steps as in previous figures), before, 3 min after addition of 20 μm DiC8 to the bath (denoted by *) and following wash-out. The pipette solution contained 24 μm of the peptide LSETKPAV.

In 16 cells in which recordings were made with the scrambled peptide, the addition of DiC8 produced a 12.9 ± 1.6 % attenuation of It, which is significantly (P < 0.001) larger than with the inhibitory peptide. Iss was attenuated by 6.6 ± 1.9 %, which is larger than with the inhibitory peptide (although for these two groups the difference for Iss did not reach statistical significance).

Hypothyroid conditions

Recordings were made from myocytes obtained from hypothyroid rats, using either the inhibitory or the control peptides in the pipette solution. DiC8 (20 μm) was added several minutes after cell rupture. Initially, the peptide inhibiting translocation of PKCε (EAVSKPLT) was used. In the presence of this peptide, the ability of DiC8 to reversibly attenuate both It and Iss was restored to a substantial (and significant) degree. An example is shown in Fig. 9.

Figure 9. Restoration of the attenuating effects of DiC8 under hypothyroid conditions by the PKCε translocation inhibitor.

Figure 9

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A, time course of changes in magnitude of It and Iss following addition of DiC8 to a cell from a hypothyroid rat. These recordings were done with EAVSKPLT (24 μm) in the pipette and show attenuation of both currents (mainly It). The effect was reversible following wash-out of DiC8 (discontinuous recording). B, superimposed current traces (same voltage steps as in previous figures, same cell as in A) obtained before and 5 min after addition of 20 μm DiC8 (denoted by *). This result indicates that the presence of this peptide enables DiC8 to attenuate the currents under hypothyroid conditions, in which the effect is usually absent.

In 31 cells (with EAVSKPLT in the pipette), DiC8 attenuated It by 11.9 ± 1.5 %, and Iss by 9.8 ± 1.5 %, which is significantly larger (P < 0.001) then the attenuation obtained in hypothyroid myocytes using the peptide-free filling solution. In four cells the effect was reversible upon wash-out. Control experiments were performed using the scrambled peptide (LSETKPAV) to ensure that there were no non-specific effects obtained by introduction of peptides under hypothyroid conditions. Under these conditions, there was no effect of DiC8 on either current, as was the case with a peptide-free filling solution. In 14 hypothyroid cells with the scrambled peptide in the pipette solution, It was attenuated by 3.3 ± 1.2 %, and Iss was attenuated by 4.7 ± 2.3 %. The summary of the effects of DiC8 on It using the different peptides in myocytes from control and hypothyroid rats is shown in Fig. 10.

Figure 10. Summary histograms showing the percentage attenuation of It by 20 μm DiC8 in control (left) and hypothyroid (right) conditions.

Figure 10

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Columns and bars indicate mean values ±s.e.m. Open columns, no peptide in the pipette solution; hatched columns, with the inhibitor of PKCε translocation EAVSKPLT (24 μm) in the pipette; cross-hatched columns, with a scrambled peptide (LSETKPAV, 24 μm) in the pipette. Whereas the inhibition of PKCε translocation blocks the action of DiC8 in normal conditions, it restores the effect of DiC8 under hypothyroid conditions. Horizontal bars and asterisks indicate significant differences between no peptide and the inhibitory peptide and between the inhibitory peptide and the scrambled peptide (P < 0.001).

DISCUSSION

Summary of results

The results presented here show for the first time that a regulatory effect of PKC activation on cardiac K+ currents (Figs 1 and 2) is significantly diminished or abolished under conditions in which PKC activity and expression have been shown to be enhanced (Figs 3 and 5). This alteration in PKC regulation of It and Iss occurs in myocytes from both hypothyroid and diabetic (insulin-deficient) rats, suggesting that this may be a general phenomenon, pertaining to the many pathological conditions in which PKC is altered. The changes in the thyroidectomized and diabetic rats were shown to be reversed by, respectively, replacement of T3 (Fig. 4) or insulin (Fig. 6). Furthermore, it was shown that a peptide inhibitor of PKCε translocation blocks the effect of a PKC activator in control cells (Fig. 7), and restores the attenuation of K+ currents by PKC activation in hypothyroid conditions (Fig. 9).

Significance and implications

The present results suggest a new mechanism by which ion channel regulation may be modified. Thus, following chronic upregulation of PKC, the attenuation of K+ currents by PKC activation is diminished or abolished. The working hypothesis is that since PKC is already upregulated, no further effects of PKC activation can be elicited. This assumption, based on the fact that PKC action is known to be regulated by translocation and binding to specific anchoring proteins (Mochly-Rosen & Gordon, 1998), was examined in two stages. First, it was shown (Fig. 6) that translocation of the major cardiac PKCε isozyme is a mediator of the action of DiC8 on the two K+ currents. This is the first finding linking a specific isozyme to PKC actions on cardiac K+ currents. Recent work has linked the β isozyme to the regulation of cardiac Ca2+ channels, also using peptides which specifically inhibit the translocation of this isozyme (Zhang et al. 1997). Our results with PKC blockers (Fig. 2), as well as the absence of an effect of DiC8 in the presence of the translocation inhibitor, rule out the possibility that DiC8 actions in these experiments involve a non-PKC-related mechanism, as suggested by Bowlby & Levitan (1995).

Secondly, it was shown that introducing the PKCε translocation inhibitor into hypothyroid cells restores the action of DiC8 (Fig. 8). This is assumed to reflect a ‘competing off’ effect whereby some of the PKC is displaced from its RACK. Based on the assumption that PKC can shuttle between different anchoring proteins which bind either inactive or active PKC (Mochly-Rosen & Gordon, 1998), the following scheme is suggested: hypothyroid conditions shift the normal distribution between PKC-RICK and PKC-RACK towards a predominance of PKC-RACK. This prevents further effects of PKC activators. Introducing a translocation inhibitor shifts the balance towards a more normal distribution, so that a subsequent addition of DiC8 can again shift the balance towards a predominantly RACK-bound state, with the resulting effect of current attenuation. Similar effects of isozyme-specific peptides have been shown to both reverse PKC binding to intracellular membranes (Blobe et al. 1996) and to reverse the effects of PKC actions (Lee et al. 1999).

Since these results were found in two different conditions of hormonal imbalance, this may reflect a common situation, applicable to other conditions in which PKC activity is altered such as in ischaemia or hypoxia (Yoshida et al. 1996; Goldberg et al. 1997). The modulation of K+ currents by agonists that act through PKC activation may be altered under such conditions. On the basis of the present findings, it is expected that the action of such agonists would be reduced when baseline PKC activity is augmented. Interestingly, in many pathological conditions, in which there is an augmented PKC activity, there is also an attenuation of K+ currents, such as occurs in hypoxia (Thierfelder et al. 1994), metabolic inhibition (Ogbaghebriel & Shrier, 1994) or hypertrophy (Meszaros et al. 1996). This is also the case in hypothyroid and diabetic conditions (Magyar et al. 1992; Jourdon & Feuvray, 1993; Shimoni et al. 1994; Shimoni & Severson, 1995). This suggests that chronic PKC upregulation may underlie the attenuation in It and Iss in these conditions.

Limitations of this study

A limitation of the present work is the lack, at this stage, of more direct evidence for the translocation of the PKCε isoform following addition of DiC8 under control conditions, and for the absence of such translocation either in hypothyroid and diabetic conditions, or in control conditions in the presenc of the inhibitory peptide. However, enhanced PKCε expression and activity have been established by others, in both models used here. This presumably results in chronic translocation of PKCε to its RACK(s), although this has not been shown directly. Thus, the present results and interpretation are consistent with a proposed change in the balance between PKC-RICKS and PKC-RACKS. A further point to be considered is that the results obtained using the peptides depend on their specificity. Although the specificity is not absolute in terms of binding to other isoforms of PKC (Lee et al. 1999), these peptides are the best tools currently available, and are far more specific than other activators or inhibitors of PKC.

Acknowledgments

This work was supported by a grant from the Alberta Heart and Stroke Foundation. I would also like to thank Dr M. P. Walsh for his advice and support, as well as for his generous gift of one of the peptides used in this study.

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