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Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels.

ABSTRACT: Potassium channels that are inhibited by internal ATP (K(ATP) channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K(ATP) channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K(ATP) channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC similarly regulates recombinant channels from both the pancreas and heart. Surprisingly, PKC acts via phosphorylation of a specific, conserved threonine residue (T180) in the pore-forming subunit (Kir6.2). Additional PKC consensus sites exist on both Kir and the larger sulfonylurea receptor (SUR) subunits. Nonetheless, T180 controls changes in open probability induced by direct PKC action either in the absence of, or in complex with, the accessory SUR1 (pancreatic) or SUR2A (cardiac) subunits. The high degree of conservation of this site among different K(ATP) channel isoforms suggests that this pathway may have wide significance for the physiological regulation of K(ATP) channels in various tissues and organelles.

SUBMITTER: Light PE

PROVIDER: S-EPMC16821 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels.

Light P E PE Bladen C C Winkfein R J RJ Walsh M P MP French R J RJ

Proceedings of the National Academy of Sciences of the United States of America 20000801 16

Potassium channels that are inhibited by internal ATP (K(ATP) channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K(ATP) channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K(ATP) channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC s ...[more]

PMID: 10908656

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Project description: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 (K(ATP)) 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 K(ATP) 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 K(ATP)) 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/H(2)O(2) scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H(2)O(2) 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 K(ATP) 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 K(ATP) channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H(2)O(2) 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 K(ATP) channels and contribute to cardiac protection against ischemia-reperfusion injury.

Project description:ATP-sensitive potassium channels (KATP channels) are hetero-octameric nucleotide-gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, KATP channels are activated during ischaemia and potentially during adrenergic stimulation. In the vasculature, they are normally active at a low level, reducing vascular tone, but the ubiquitous nature of these channels leads to complex and poorly understood channelopathies as a result of gain- or loss-of-function mutations. Zebrafish (ZF) models of these channelopathies may provide insights to the link between molecular dysfunction and complex pathophysiology, but this requires understanding the tissue dependence of channel activity and subunit specificity. Thus far, direct analysis of ZF KATP expression and functional properties has only been performed in pancreatic β-cells. Using a comprehensive combination of genetically modified fish, electrophysiology and gene expression analysis, we demonstrate that ZF cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular KATP channels, ZF channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. The results provide a first characterization of the molecular properties of fish KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome. KEY POINTS: Zebrafish cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular KATP channels, zebrafish channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. We provide a first characterization of the molecular properties of fish KATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome.

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Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels.

Publications

Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels.

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