Mechanisms of IhERG/IKr Modulation by α1-Adrenoceptors in HEK293 Cells and Cardiac Myocytes

Abstract

Background: The rapid delayed rectifier K⁺ current (I_Kr), carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR) activation reduces I_Kr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates I_Kr. Methods: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP₂-interacting mutant D-hERG (with a deletion of the 883-894 amino acids) in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG) were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. I_Kr was recorded in isolated feline cardiac myocytes. Results: Activation of the α1-AR reduces the amplitude of I_hERG and I_Kr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP₂ hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated I_Kr channel phosphorylation and subsequent I_Kr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. Conclusions: These findings clarify the link between sympathetic nervous system hyperactivity and I_Kr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G‐protein coupled receptors

“…Moreover, we found that A61603 produced a similar effect to the cPKC‐specific activator peptide. The results are consistent with the previous reports that PKC activators or phenylephrine cause a depolarizing shift in the voltage‐dependent activation of the hERG channels (Thomas et al, , ; Urrutia et al, ). Western blot analysis showed that the phospho‐PKCα, but not phospho‐PKCε, was increased in the ventricular myocytes after α 1A ‐adrenoreceptor stimulation.…”

“…Our previous study has shown that stimulation of AT 1 receptors produces an acute inhibitory effect on I Kr /hERG currents via a PKC pathway in guinea pig ventricular myocytes and HEK293 cells (Wang et al, ). Meanwhile, activation of α 1A ‐adrenoceptors also results in an instant inhibition of I Kr /hERG current through PKC signalling (Thomas et al, ; Urrutia et al, ). Accordingly, hERG channels represent a target for modulation by autonomic neurotransmitters and hormones, and thus, PKC may become a candidate mediator of the pathological cardiac electrophysiological remodelling.…”

“…However, it has been noted that the modulation of the hERG channels by these two GPCRs shows a different mode of action. The activation of α 1A ‐adrenoreceptors shifts the voltage‐dependent activation curve to the right (Thomas et al, ; Urrutia et al, ), whereas AT 1 receptor agonists do not (Wang et al, ). It is known that PKCs can be classified into three subgroups based on their structure and activation requirements.…”

PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

“…They also confirm a role of the N-terminal part of the protein as proposed by other studies [11,34]. Cockerill and coworkers found that residues 2-354 of the K V 11.1 N-terminus are critical for both cPKC phosphorylation and functional modulation of the channel [11].…”

Synergistic Inhibition of Delayed Rectifier K+ and Voltage-Gated Na+ Currents by Artemisinin in Pituitary Tumor (GH3) Cells

“…The pharmacological and biophysical properties of I K(erg) in GH 3 cells were previously described [19]. Physiological roles of I K(erg) in different types of cells have been also reported [20,21]. However, at the level of -100 mV, the peak amplitude of deactivating I K(erg) did not differ significantly between the absence and presence of 10 μM ART (787 ± 14 pA [control] versus 782 ± 13 pA [in the presence of ART], n = 7, P > 0.05).…”