Search

To elucidate the ionic mechanism of endothelin-1 (ET-1)-induced focal ventricular tachyarrhythmias, the regulation of I(K1) and its main molecular correlates, Kir2.1, Kir2.2 and Kir2.3 channels, by ET-1 was investigated. Native I(K1) in human atrial cardiomyocytes was studied with whole-cell patch clamp. Human endothelin receptors were coexpressed with human Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes. Currents were measured with a two-microelectrode voltage clamp. In human cardiomyocytes, ET-1 induced a marked inhibition of I(K1) that could be suppressed by the protein kinase C (PKC) inhibitor staurosporine. To investigate the molecular mechanisms underlying this regulation, we studied the coupling of ET(A) receptors to homomeric and heteromeric Kir2.1, Kir2.2 and Kir2.3 channels in the Xenopus oocyte expression system. ET(A) receptors coupled functionally to Kir2.2 and Kir2.3 channels but not to Kir2.1 channels. In Kir2.2 channels lacking functional PKC phosphorylation sites, the inhibitory effect was abolished. The inhibition of Kir2.3 currents could be suppressed by the PKC inhibitors staurosporine and chelerythrine. The coupling of ET(A) receptors to heteromeric Kir2.1/Kir2.2 and Kir2.2/Kir2.3 channels resulted in a strong inhibition of currents comparable with the effect observed in Kir2.2 homomers. Surprisingly, in heteromeric Kir2.1/Kir2.3 channels, no effect was observed. ET-1 inhibits human cardiac I(K1) current via a PKC-mediated phosphorylation of Kir2.2 channel subunits and additional regulatory effects on Kir2.3 channels. This mechanism may contribute to the intrinsic arrhythmogenic potential of ET-1.

Objective: Hereditary long QT syndrome (LQTS) is a genetically heterogeneous disease characterized by prolonged QT intervals and an increased risk for ventricular arrhythmias and sudden cardiac death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of LQTS. KCNQ1 is associated with two different entities of LQTS, the autosomal-dominant Romano-Ward syndrome (RWS), and the autosomal-recessive Jervell and Lange-Nielsen syndrome (JLNS) characterized by bilateral deafness in addition to cardiac arrhythmias. In this study, we investigate and discuss dominant-negative I(Ks) current reduction by a KCNQ1 deletion mutation identified in a RWS family., Methods: Single-strand conformation polymorphism analysis and direct sequencing were used to screen LQTS genes for mutations. Mutant KCNQ1 channels were heterologously expressed in Xenopus oocytes, and potassium currents were recorded using the two-microelectrode voltage clamp technique., Results: A heterozygous deletion of three nucleotides (CTT) identified in the KCNQ1 gene caused the loss of a single phenylalanine residue at position 339 (KCNQ1-deltaF339). Electrophysiological measurements in the presence and absence of the regulatory beta-subunit KCNE1 revealed that mutant and wild type forms of an N-terminal truncated KCNQ1 subunit (isoform 2) caused much stronger dominant-negative current reduction than the mutant form of the full-length KCNQ1 subunit (isoform 1)., Conclusion: This study highlights the functional relevance of the truncated KCNQ1 splice variant (isoform 2) in establishment and mode of inheritance in long QT syndrome. In the RWS family presented here, the autosomal-dominant trait is caused by multiple dominant-negative effects provoked by heteromultimeric channels formed by wild type and mutant KCNQ1-isoforms in combination with KCNE1.

Patients with cardiac disease typically develop life-threatening ventricular arrhythmias during physical or emotional stress, suggesting a link between adrenergic stimulation and regulation of the cardiac action potential. Human ether-a-go-go related gene (hERG) potassium channels conduct the rapid component of the repolarizing delayed rectifier potassium current, I(Kr). Previous studies have revealed that hERG channel activation is modulated by activation of the beta-adrenergic system. In contrast, the influence of the alpha-adrenergic signal transduction cascade on hERG currents is less well understood. The present study examined the regulation of hERG currents by alpha(1A)-adrenoceptors. hERG channels and human alpha(1A)-adrenoceptors were heterologously coexpressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. Stimulation of alpha(1A)-receptors by applying 20 microM phenylephrine caused hERG current reduction due to a 9.6-mV shift of the activation curve towards more positive potentials. Simultaneous application of the alpha(1)-adrenoceptor antagonist prazosin (20 microM) prevented the activation shift. Inhibition of PKC (3 microM Ro-32-0432) or PKA (2.5 microM KT 5720) abolished the alpha-adrenergic activation shift, suggesting that PKC and PKA are required within the regulatory mechanism. The effect was still present when the PKA- and PKC-dependent phosphorylation sites in hERG were deleted by mutagenesis. In summary, cardiac repolarizing hERG/I(Kr) potassium currents are modulated by alpha(1A)-adrenoceptors via PKC and PKA independently of direct channel phosphorylation. This novel regulatory pathway of alpha1-adrenergic hERG current regulation provides a link between stress and ventricular arrhythmias, in particular in patients with heart disease.

Objective: The cardiac inwardly rectifying potassium current IK1 and its molecular correlates Kir2.1 and Kir2.2 play an important role in cardiac repolarisation and in the pathogenesis of hereditary long-QT syndrome (LQTS-7). Protein kinases A (PKA) and C (PKC) are key enzymes in adrenergic signal transduction, inducing arrhythmias in heart disease. This study investigated the regulation of Kir2.2 (KCNJ12) by PKA., Methods: Cloned Kir2.2 channels were expressed heterologously in Xenopus oocytes and currents were measured with the double-electrode voltage-clamp technique., Results: After activation of PKA by forskolin (100 micromol/l) or Ro-20-1724 (100 micromol/l), wild type currents at -120 mV were increased by 93.7% and 79.0%, respectively. Coapplication of the PKA inhibitor KT-5720 (2.5 micromol/l) attenuated this effect. No significant changes were apparent after mutation of the single PKA consensus site S430. In addition, removal of all four PKC consensus sites in Kir2.2 induced a phorbolester-mediated current increase which could be suppressed by PKA inhibitors H-89 (50 micromol/l) and KT-5720 (2.5 micromol/l)., Conclusions: This study demonstrates antagonistic effects of PKA and PKC in the regulation of Kir2.2. Phosphorylation by PKC has been shown to cause an inhibition of Kir2.2 currents, whereas activation of PKA leads to current upregulation., (Copryright 2004 European Society of Cardiology)

1 The topoisomerase II inhibitor amsacrine is used in the treatment of acute myelogenous leukemia. Although most anticancer drugs are believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by reports of QT interval prolongation, ventricular fibrillation and death associated with amsacrine treatment. Since blockade of cardiac human ether-a-go-go-related gene (HERG) potassium currents is an important cause of acquired LQTS, we investigated the acute effects of amsacrine on cloned HERG channels to determine the electrophysiological basis for its proarrhythmic potential. 2 HERG channels were heterologously expressed in human HEK 293 cells and Xenopus laevis oocytes, and the respective potassium currents were recorded using patch-clamp and two-microelectrode voltage-clamp electrophysiology. 3 Amsacrine blocked HERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner, with IC50 values of 209.4 nm and 2.0 microm, respectively. 4 HERG channels were primarily blocked in the open and inactivated states, and no additional voltage dependence was observed. Amsacrine caused a negative shift in the voltage dependence of both activation (-7.6 mV) and inactivation (-7.6 mV). HERG current block by amsacrine was not frequency dependent. 5 The S6 domain mutations Y652A and F656A attenuated (Y652A) or abolished (F656A, Y652A/F656A) HERG current blockade, indicating that amsacrine binding requires a common drug receptor within the pore-S6 region. 6 In conclusion, these data demonstrate that the anticancer drug amsacrine is an antagonist of cloned HERG potassium channels, providing a molecular mechanism for the previously reported QTc interval prolongation during clinical administration of amsacrine.

Objective: Patients with HERG-associated long QT syndrome typically develop tachyarrhythmias during physical or emotional stress. Previous studies have revealed that activation of the beta-adrenergic system and consecutive elevation of the intracellular cAMP concentration regulate HERG channels via protein kinase A-mediated phosphorylation of the channel protein and via direct interaction with the cAMP binding site of HERG. In contrast, the influence of the alpha-adrenergic signal transduction cascade on HERG currents as suggested by recent reports is less well understood. The aim of the present study was to elucidate the biochemical pathways of the protein kinase C (PKC)-dependent regulation of HERG currents., Methods: HERG channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique., Results: Application of the phorbol ester PMA, an unspecific protein kinase activator, shifted the voltage dependence of HERG activation towards more positive potentials. This effect could be mimicked by activation of conventional PKC isoforms with thymeleatoxin. Coexpression of HERG with the beta-subunits minK or hMiRP1 did not alter the effect of PMA. Specific inhibition of PKC abolished the PMA-induced activation shift, suggesting that PKC is required within the regulatory mechanism. The PMA-induced effect could still be observed when the PKC-dependent phosphorylation sites in HERG were deleted by mutagenesis. Cytoskeletal proteins such as actin filaments or microtubules did not affect the HERG activation shift., Conclusion: In addition to the known effects of PKA and cAMP, HERG channels are also modulated by PKC. The molecular mechanisms of this PKC-dependent process are not completely understood but do not depend on direct PKC-dependent phosphorylation of the channel.

(1) Acquired long QT syndrome (aLQTS) is caused by prolongation of the cardiac action potential because of blockade of cardiac ion channels and delayed repolarization of the heart. Patients with aLQTS carry an increased risk for torsade de pointes arrhythmias and sudden cardiac death. Several antipsychotic drugs may cause aLQTS. Recently, cases of QTc prolongation and torsade de pointes associated with chlorpromazine treatment have been reported. Blockade of human ether-a-go-go-related gene (HERG) potassium channels, which plays a central role in arrhythmogenesis, has previously been reported to occur with chlorpromazine, but information on the mechanism of block is currently not available. We investigated the effects of chlorpromazine on cloned HERG potassium channels to determine the biophysical mechanism of block. (2) HERG channels were heterologously expressed in Xenopus laevis oocytes, and ion currents were measured using the two-microelectrode voltage-clamp technique. (3) Chlorpromazine blocked HERG potassium channels with an IC(50) value of 21.6 micro M and a Hill coefficient of 1.11. (4) Analysis of the voltage dependence of block revealed a reduction of inhibition at positive membrane potentials. (5) Inhibition of HERG channels by chlorpromazine displayed reverse frequency dependence, that is, the amount of block was lower at higher stimulation rates. No marked changes in electrophysiological parameters such as voltage dependence of activation or inactivation, or changes of the inactivation time constant were observed. (6) In conclusion, HERG channels were blocked in the closed and activated states, and unblocking occurred very slowly.

Background: Protein kinases A (PKA) and C (PKC) are activated in ischemic preconditioning and heart failure, conditions in which patients develop arrhythmias. The native inward rectifier potassium current (IK1) plays a central role in the stabilization of the resting membrane potential and the process of arrhythmogenesis. This study investigates the functional relationship between PKC and IK1., Methods and Results: In whole-cell patch-clamp experiments with isolated human atrial cardiomyocytes, the IK1 was reduced by 41% when the nonspecific activator of PKC phorbol 12 myristate 13-acetate (PMA; 100 nmol/L) was applied. To investigate the effects of PKC on cloned channel underlying parts of the native IK1, we expressed Kir(2.1b) heterologously in Xenopus oocytes and measured currents with the double-electrode voltage-clamp technique. PMA decreased the current by an average of 68%, with an IC50 of 0.68 nmol/L. The inactive compound 4-alpha-PMA was ineffective. Thymeleatoxin and 1-oleolyl-2-acetyl-sn-glycerol, 2 specific activators of PKC, produced effects similar to those of PMA. Inhibitors of PKC, ie, staurosporine and chelerytrine, could inhibit the PMA effect (1 nmol/L) significantly. After mutation of the PKC phosphorylation sites (especially S64A and T353A), PMA became ineffective., Conclusions: The human IK1 in atrial cardiomyocytes and one of its underlying ion channels, the Kir(2.1b) channel, is inhibited by PKC-dependent signal transduction pathways, possibly contributing to arrhythmogenesis in patients with structural heart disease in which PKC is activated.

1. Bertosamil is chemically related to the class-III anti-arrhythmic drug tedisamil and has been developed as a bradycardic, anti-ischemic and anti-arrhythmic drug. Its anti-arrhythmic properties might in part be attributed to its block of voltage-dependent potassium channels Kv(1.2), Kv(1.4). and Kv(1.5). However, HERG-potassium channel block as an important target for class-III drugs has not yet been investigated. 2. We investigated the effect of bertosamil on the HERG potassium channel heterologously expressed in Xenopus oocytes with the two-electrode voltage-clamp technique. 3. Bertosamil (70 microM) inhibited HERG tail currrent after a test pulse to 30 mV by 49.3+/-8.4% (n=5) and the IC(50) was 62.7 microM. Onset of block was fast, i.e. 90% of inhibition developed within 180+/-8.22 s (n=5), and block was totally reversible upon washout within 294+/-38.7 s (n=5). 4. Bertosamil-induced block of HERG potassium channels was state-dependent with block mainly to open- and inactivated channels. Half-maximal activation voltage was slightly shifted towards more negative potentials. 5. Steady-state inactivation of HERG was not influenced by bertosamil. Bertosamil block elicited voltage-but no frequency-dependent effects. 6. In summary, bertosamil blocked the HERG potassium channel. These blocking properties may contribute to the anti-arrhythmic effects of bertosamil in the treatment of atrial and particular ventricular arrhythmias.

Objective: To investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line., Methods: HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody., Results: Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated., Conclusions: PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.

Objective: The antiarrhythmic potential of betablockers contributes to their beneficial effects in the treatment of cardiac diseases, although the molecular basis of their class II antiarrhythmic action has not been clarified yet., Methods: To investigate a putative functional link between beta-adrenoreceptors and the fast component of cardiac delayed rectifier K(+) channels (I(Kr)), whole-cell patch-clamp experiments were performed with isolated guinea-pig ventricular myocytes. Tail currents of I(Kr) were measured at -40 mV after short (200 ms) test pulses to +40 mV., Results: After application of the unspecific beta-receptor agonist isoproterenol (10 microM) for 12 min, the I(Kr) tail current was decreased by 72%, with an IC(50) of 1.4 microM. The specific beta(1)-blocker CGP207120A (10 microM) significantly attenuated the isoproterenol effect (net 24% decrease). The specific beta(1)-agonist xamoterol (10 microM), could mimic the isoproterenol effect (58% decrease). Modulators of beta(2)- or beta(3)-adrenoreceptors were far less effective. When isoproterenol or xamoterol were combined with KT5720 (2.5 microM), a specific inhibitor of protein kinase A (PKA), their effects were drastically reduced, indicating that PKA presumably mediates the beta(1)-adrenergic inhibition of I(Kr). Tail current reductions by cAMP, forskolin, PKA catalytic subunit and a combination of PKA holoenzyme and cAMP support an involvement of PKA in the regulation of I(Kr)., Conclusions: The functional link between I(Kr) and the beta(1)-adrenergic receptor involving PKA may play an important role in arrhythmogenesis and contribute to the antiarrhythmic action of clinically used beta(1)-blockers.

Objective: The aryloxypropanolamine carvedilol is a multiple action cardiovascular drug with blocking effects on alpha-receptors, beta-receptors, Ca(2+)-channels, Na(+)-channels and various native cardiac K(+) channels, thereby prolonging the cardiac action potential. In a number of clinical trials with patients suffering from congestive heart failure, carvedilol appeared to be superior to other beta-blocking agents in reducing total mortality. Given the multiple pharmacological actions of carvedilol, this may be due to specific channel blockade rather than beta-antagonistic activity. Since human ether-a-go-go related gene (HERG) K(+)channels play a critical role in the pathogenesis of cardiac arrhythmias and sudden cardiac death, the effects of carvedilol on HERG K(+)channels were investigated., Methods: Double-electrode voltage-clamp experiments were performed on HERG potassium channels which were expressed heterologously in Xenopus oocytes., Results: Carvedilol at a concentration of 10 microM blocked HERG potassium tail currents by 47%. The electrophysiological characteristics of HERG, i.e. activation, steady-state inactivation and recovery from inactivation were not affected by carvedilol. Inhibition of current gradually increased from 0% immediately after the test pulse to about 80% at 600 ms with subsequent marginal changes of current kinetics during the resting 29 s, indicating a very fast open channel block by carvedilol as the major blocking mechanism., Conclusion: This is the first study demonstrating that carvedilol blocks HERG potassium channels. The biophysical data presented in this study with a potentially antiarrhythmic effect may contribute to the positive outcome of clinical trials with carvedilol.