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Oxidation of cysteine 117 stimulates constitutive activation of the type I? cGMP-dependent protein kinase.

ABSTRACT: The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type I? isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG I?. PKG I? C42S and a truncation construct that does not contain Cys-42 (?53) were both constitutively activated by H2O2 In contrast, oxidation of PKG I? C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG I? C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG I?-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG I? activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG I? C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG I? C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG I? C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG I?. Cumulatively, these results indicate that constitutive activation of PKG I? proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

SUBMITTER: Sheehe JL

PROVIDER: S-EPMC6204908 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase.

Sheehe Jessica L JL Bonev Adrian D AD Schmoker Anna M AM Ballif Bryan A BA Nelson Mark T MT Moon Thomas M TM Dostmann Wolfgang R WR

The Journal of biological chemistry 20180911 43

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an <i>in vitro</i> assay, we observed that oxidat ...[more]

PMID: 30206122

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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.

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Oxidation of cysteine 117 stimulates constitutive activation of the type I? cGMP-dependent protein kinase.

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Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase.

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