Abstract
We investigated the effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on the rapid component of the delayed rectifier potassium current, IKr, in guinea pig cardiomyocytes and found that the IKr current amplitude was reduced by 20% with 10 nM PMA and 44% with 100 nM PMA. The ether-a-go-go-related gene (HERG) encodes IKr in human heart. We expressed HERG heterologously in Xenopus oocytes and investigated the effects of PMA on the delayed rectifier potassium current. Upon application of PMA in a concentration of 100 nM, we found a similar reduction of HERG outward current amplitude by 59%. This reduction was due to a shift in the HERG activation curve by 37 mV. The ED50 for the PMA-induced shift was 9.0 nM. The inactive 4alpha-phorbol 12-myristate 13-acetate (4alpha-PMA) had no effect. PMA is known to act by stimulating distinct protein kinase cascades. Additional application of the specific protein kinase C inhibitors chelerythrine (10 microM) or bisindolylmaleimide (1 microM) could not attenuate the PMA-induced shift. In contrast, the shift by PMA was reduced significantly when the specific protein kinase A (PKA) inhibitors H89 (50 microM) or KT5720 (2.5 microM) were applied. Forskolin (400 microM), an activator of the adenylate cyclase that results in PKA activation, shifted the HERG activation curve by 14 mV. Moreover the specific protein kinase C activator 1-stearoyl-2-arachidonylglycerol (10 microM) showed no effect. Our data suggest that mainly PKA is mediating the shift of the HERG activation kinetics.
Highlights
Repolarization of the cardiac action potential involves many potassium currents [1]
We investigated the effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on the rapid component of the delayed rectifier potassium current, IKr, in guinea pig cardiomyocytes and found that the IKr current amplitude was reduced by 20% with 10 nM PMA and 44% with 100 nM PMA
Using combinations of various activators and inhibitors, we provide evidence that protein kinase A (PKA) is a key enzyme involved in the PMA-induced shift of the human ether-a-go-gorelated gene (HERG) activation kinetics
Summary
Repolarization of the cardiac action potential involves many potassium currents [1]. One crucial repolarizing potassium current is the rapid component of the delayed rectifier potassium current (IKr)1 [2,3,4]. HERG expressed in Xenopus oocytes produces a potassium current largely indistinguishable from native cardiac IKr [6, 7]. Electrophysiological studies have shown that these HERG mutations result in a reduced HERG current with the consequence that the cardiac action potential and QT interval on the surface electrocardiogram of this patients is prolonged [10]. The aim of this study was to investigate whether protein kinases, PKA and PKC, are involved in the regulation of the HERG potassium channel, which underlies IKr. Because protein kinases are present endogenously in Xenopus oocytes, we could use protein kinase activators and inhibitors. Using combinations of various activators and inhibitors, we provide evidence that PKA is a key enzyme involved in the PMA-induced shift of the HERG activation kinetics
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