Abstract
BackgroundCyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.Methods and FindingsSingle-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.ConclusionThe present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening KATP channels and contribute to cardiac protection against ischemia-reperfusion injury.
Highlights
The ATP-sensitive potassium (KATP) channel functions as a high-fidelity metabolic sensor which couples intracellular metabolic state to membrane excitability [1,2,3]
The present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by reactive oxygen species (ROS) (H2O2 in particular), calmodulin and calmodulin-dependent protein kinase II (CaMKII), alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins
Our earlier study unravels that PKG bidirectionally regulates the function of neuronal KATP (i.e., Kir6.2/SUR1) channels, encompassing a predominating stimulatory action, which can be reproduced by nitric oxide (NO) via activation of a Cyclic GMP (cGMP)/soluble guanylyl cyclase/PKG signaling cascade, and a moderate inhibitory action, which likely involves direct PKG phosphorylation of the channel or some closely associated regulatory protein(s) [24]; our findings suggest that the stimulatory action of PKG on neuronal KATP channels is mediated by intracellular signaling through the 5-hydroxydecanoate (5-HD)-sensitive factor, reactive oxygen species (ROS), calcium, and calmodulin [25]
Summary
The ATP-sensitive potassium (KATP) channel functions as a high-fidelity metabolic sensor which couples intracellular metabolic state to membrane excitability [1,2,3]. In cardiac and skeletal muscles KATP channels are composed of Kir6.2 and SUR2A subunits [9,10], whereas in central neurons and pancreatic b-cells they are composed of Kir6.2 and SUR1 subunits [11]. KATP channels are widely expressed in excitable tissues and serve a variety of important cellular functions, including glucose-stimulated insulin secretion, neurotransmitter release, vascular tone, and protection of neurons and cardiomyocytes under metabolic stress [12]. Functional modulation of KATP channels by cGMP, presumably through activation of PKG, has been demonstrated in vascular smooth muscle cells [22]. Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; the underlying mechanism remains largely unknown.
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