Activation of Protein Kinase C ϵ Inhibits the Two-pore Domain K+ Channel, TASK-1, Inducing Repolarization Abnormalities in Cardiac Ventricular Myocytes

Abstract

Activation of the platelet-activating factor (PAF) receptor leads to a decrease in outward current in murine ventricular myocytes by inhibiting the TASK-1 channel. TASK-1 carries a background or "leak" current and is a member of the two-pore domain potassium channel family. Its inhibition is sufficient to delay repolarization, causing prolongation of the action potential duration, and in some cases, early after depolarizations. We set out to determine the cellular mechanisms that control regulation of TASK-1 by PAF. Inhibition of TASK-1 via activation of the PAF receptor is protein kinase C (PKC)-dependent. Using isoform-specific PKC inhibitor or activator peptides in patch clamp experiments, we now demonstrate that activation of PKC⑀ is both necessary and sufficient to regulate murine TASK-1 current in a heterologous expression system and to induce repolarization abnormalities in isolated myocytes. Furthermore, site-directed mutagenesis studies have identified threonine 381, in the C-terminal tail of murine TASK-1, as a critical residue in this regulation.Regulation of cardiac function depends on the appropriate cumulative activity of numerous ion channels in individual cardiomyocytes that are responsible for the sequential depolarization-repolarization cycle known as the action potential (AP). 1 Although the major currents contributing to the AP have been described (1), additional small currents at specific points of low conductance in the AP cycle are sufficient to induce repolarization abnormalities in isolated cells (2, 3) and therefore arrhythmias in situ. These arrhythmias may contribute to the electrical abnormalities that lead to sudden death after myocardial infarction, which persists as the number one cause of death in the United States. We have focused on one channel that has been proposed to contribute to cardiac arrhythmias, TASK-1, a member of the recently described family of two-pore domain potassium channels (4).The two-pore domain K channel family is composed of at least 15 different members. These channels are widely distributed in excitable tissue, primarily in the brain and heart, and in general are responsive to environmental cues such as temperature, pH, and stretch (5, 6). Several are also regulated by lipids such as arachidonic acid or platelet-activating factor (PAF) (7-9). PAF is an inflammatory phospholipid that has been linked to arrhythmogensis in isolated canine ventricular myocytes (10). We have recently shown that PAF regulates the TASK-1 channel and determined that the arrhythmogenic effect of the stable PAF analog, carbamyl-platelet-activating factor (C-PAF) in mouse cardiomyocytes, is due to the inhibition of TASK-1 current in a protein kinase C (PKC)-dependent manner (2).In this study, we elucidate the molecular mechanism of the C-PAF effect on TASK-1 current by identifying the ⑀ isoform of PKC (PKC⑀) as a critical component in PAFR signaling. In addition, using site-directed mutagenesis, we have tentatively identified the critical residue that is the target for PKC in the mu...

Ability to Induce Atrial Fibrillation in the Peri-operative Period Is Associated with Phosphorylation-dependent Inhibition of TWIK Protein-related Acid-sensitive Potassium Channel 1 (TASK-1)

“…4B; p Ͻ 0.01). To verify the requirement for PKC⑀ in the C-PAF-induced inhibition of the TASK-1 current, in agreement with previous observations in the murine channel, a specific PKC⑀-inhibitor peptide was used (7,18). Addition of this peptide but not a scrambled control peptide to the patch pipette (100 nM) blocked the effect of C-PAF on all previously responsive forms of the channel ( Fig.…”

“…It has long been known that activated neutrophils contribute to arrhythmogenicity in isolated hearts, and numerous clinical studies have found a link between inflammatory mediators and arrhythmias. We have described repolarization abnormalities caused by the inflammatory mediator PAF, released from activated neutrophils (5), and we determined that C-PAF elicits these effects in vitro via PKC⑀-dependent inhibition of the TASK-1 channel in murine ventricular myocytes (6) and in heterologous expression systems (7). We have now demonstrated a similar phenomenon in intact canine atrium and associated with peri-op AF in the dog.…”

“…BIM-1 (Sigma) was dissolved in DMSO and diluted to 5 mM in incubation buffer prior to use (final DMSO Ͻ0.1%). The specific PKC⑀-inhibitor peptide and a scrambled control peptide (7,18) were synthesized by the Columbia University Medical Center Protein Core facility and were prepared in water and then diluted to the final concentration of 100 nM in pipette solution.…”

“…Activation of neutrophils alone is known to be arrhythmogenic (3,4), and one of the neutrophil products that contributes to arrhythmogenicity is the inflammatory lipid mediator, platelet-activating factor (PAF) (5). Myocytes express the PAF receptor that, when activated, leads to the activation of a downstream kinase, which we have shown targets the two-pore domain K ϩ channel (K 2P ) TASK-1, resulting in inhibition of the channel (6,7). Our current results suggest that the loss of the TASK-1 current may contribute to the arrhythmogenic effect of PAF.…”

“…Thr-383 Is an Inhibitory PKC⑀ Phosphorylation Site in the TASK-1 C Terminus-C-PAF, a nonhydrolyzable analog of the lipid inflammatory mediator, PAF, inhibits mouse TASK-1 in a PKC⑀-dependent manner by stimulating the phosphorylation of threonine 381 (Thr-381) in the murine TASK-1 C terminus (7). Therefore, we asked whether PKC⑀ directly phosphorylates canine TASK-1, and if this phosphorylation event is also associated with the loss of TASK-1 current in our peri-operative AF model.…”