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Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade.

Chai Y, Zhang DM, Lin YF - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Physiology and Membrane Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT

Background: 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 (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 findings: Single-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.

Conclusion: The 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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Roles of calmodulin and CaMKII in mediating PKG stimulation of Kir6.2/SUR2A channels.Recombinant Kir6.2/SUR2A channels were expressed in HEK293 cells by transient transfection. Cell-attached patch-clamp recordings and drug application were administered as described in Fig. 1. (A–B) Single-channel current traces of Kir6.2/SUR2A channel obtained from a cell-attached patch prior to (upper panel) and during (lower panel) application of the cGMP-specific PDE inhibitor zaprinast (50 µM) together with the membrane-permeable, selective calmodulin antagonist SKF-7171A (10 µM) (A), or with the myristoylated autocamtide-2 related inhibitory peptide for CaMKII (mAIP; 1 µM) (B). The scale bars are the same as described in Fig. 1. (C) Normalized NPo of Kir6.2/SUR2A channels obtained during application of drugs in individual groups. The zaprinast data (filled bar) are the same as presented in Fig. 1D, and are included here for comparison purpose. NPo values were normalized to the corresponding control (taken as 1) as described in Fig. 1D. The dashed line indicates the control level. Data are presented as mean ± SEM of 8–12 patches. The significance level is: **, P<0.01 (two-tailed one-sample t tests within individual groups, or Dunnett's multiple comparison tests between groups).
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pone-0018191-g004: Roles of calmodulin and CaMKII in mediating PKG stimulation of Kir6.2/SUR2A channels.Recombinant Kir6.2/SUR2A channels were expressed in HEK293 cells by transient transfection. Cell-attached patch-clamp recordings and drug application were administered as described in Fig. 1. (A–B) Single-channel current traces of Kir6.2/SUR2A channel obtained from a cell-attached patch prior to (upper panel) and during (lower panel) application of the cGMP-specific PDE inhibitor zaprinast (50 µM) together with the membrane-permeable, selective calmodulin antagonist SKF-7171A (10 µM) (A), or with the myristoylated autocamtide-2 related inhibitory peptide for CaMKII (mAIP; 1 µM) (B). The scale bars are the same as described in Fig. 1. (C) Normalized NPo of Kir6.2/SUR2A channels obtained during application of drugs in individual groups. The zaprinast data (filled bar) are the same as presented in Fig. 1D, and are included here for comparison purpose. NPo values were normalized to the corresponding control (taken as 1) as described in Fig. 1D. The dashed line indicates the control level. Data are presented as mean ± SEM of 8–12 patches. The significance level is: **, P<0.01 (two-tailed one-sample t tests within individual groups, or Dunnett's multiple comparison tests between groups).

Mentions: Our recent study suggests that calmodulin mediates PKG stimulation of neuronal KATP channels in intact cells [25]. Whether calmodulin is required for the functional effect of PKG activation on cardiac-type KATP channels is not known. To examine this possibility, SKF-7171A (10 µM), a cell-permeable calmodulin antagonist, was coapplied together with the PKG activator zaprinast (50 µM) to Kir6.2/SUR2A channels in cell-attached patches obtained from transfected HEK293 cells, following a 15-min pretreatment with SKF-7171A (10 µM). In the presence of SKF-7171A, zaprinast failed to increase the single-channel activity of Kir6.2/SUR2A channels in intact cells (Fig. 4A), resulting in an averaged, normalized NPo of 1.23±0.23 (control as 1) (Fig. 4C, open bar; 8 patches; no significant change, one-sample t test). The positive zaprinast effect on the normalized NPo of Kir6.2/SUR2A channels was completely abolished by SKF-7171A (Fig. 4C, filled vs. open bars; P<0.01, Dunnett's multiple comparison test following one-way ANOVA), indicating that the activity of calmodulin was required for PKG stimulation of cardiac-type KATP channels in intact cells.


Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade.

Chai Y, Zhang DM, Lin YF - PLoS ONE (2011)

Roles of calmodulin and CaMKII in mediating PKG stimulation of Kir6.2/SUR2A channels.Recombinant Kir6.2/SUR2A channels were expressed in HEK293 cells by transient transfection. Cell-attached patch-clamp recordings and drug application were administered as described in Fig. 1. (A–B) Single-channel current traces of Kir6.2/SUR2A channel obtained from a cell-attached patch prior to (upper panel) and during (lower panel) application of the cGMP-specific PDE inhibitor zaprinast (50 µM) together with the membrane-permeable, selective calmodulin antagonist SKF-7171A (10 µM) (A), or with the myristoylated autocamtide-2 related inhibitory peptide for CaMKII (mAIP; 1 µM) (B). The scale bars are the same as described in Fig. 1. (C) Normalized NPo of Kir6.2/SUR2A channels obtained during application of drugs in individual groups. The zaprinast data (filled bar) are the same as presented in Fig. 1D, and are included here for comparison purpose. NPo values were normalized to the corresponding control (taken as 1) as described in Fig. 1D. The dashed line indicates the control level. Data are presented as mean ± SEM of 8–12 patches. The significance level is: **, P<0.01 (two-tailed one-sample t tests within individual groups, or Dunnett's multiple comparison tests between groups).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3066208&req=5

pone-0018191-g004: Roles of calmodulin and CaMKII in mediating PKG stimulation of Kir6.2/SUR2A channels.Recombinant Kir6.2/SUR2A channels were expressed in HEK293 cells by transient transfection. Cell-attached patch-clamp recordings and drug application were administered as described in Fig. 1. (A–B) Single-channel current traces of Kir6.2/SUR2A channel obtained from a cell-attached patch prior to (upper panel) and during (lower panel) application of the cGMP-specific PDE inhibitor zaprinast (50 µM) together with the membrane-permeable, selective calmodulin antagonist SKF-7171A (10 µM) (A), or with the myristoylated autocamtide-2 related inhibitory peptide for CaMKII (mAIP; 1 µM) (B). The scale bars are the same as described in Fig. 1. (C) Normalized NPo of Kir6.2/SUR2A channels obtained during application of drugs in individual groups. The zaprinast data (filled bar) are the same as presented in Fig. 1D, and are included here for comparison purpose. NPo values were normalized to the corresponding control (taken as 1) as described in Fig. 1D. The dashed line indicates the control level. Data are presented as mean ± SEM of 8–12 patches. The significance level is: **, P<0.01 (two-tailed one-sample t tests within individual groups, or Dunnett's multiple comparison tests between groups).
Mentions: Our recent study suggests that calmodulin mediates PKG stimulation of neuronal KATP channels in intact cells [25]. Whether calmodulin is required for the functional effect of PKG activation on cardiac-type KATP channels is not known. To examine this possibility, SKF-7171A (10 µM), a cell-permeable calmodulin antagonist, was coapplied together with the PKG activator zaprinast (50 µM) to Kir6.2/SUR2A channels in cell-attached patches obtained from transfected HEK293 cells, following a 15-min pretreatment with SKF-7171A (10 µM). In the presence of SKF-7171A, zaprinast failed to increase the single-channel activity of Kir6.2/SUR2A channels in intact cells (Fig. 4A), resulting in an averaged, normalized NPo of 1.23±0.23 (control as 1) (Fig. 4C, open bar; 8 patches; no significant change, one-sample t test). The positive zaprinast effect on the normalized NPo of Kir6.2/SUR2A channels was completely abolished by SKF-7171A (Fig. 4C, filled vs. open bars; P<0.01, Dunnett's multiple comparison test following one-way ANOVA), indicating that the activity of calmodulin was required for PKG stimulation of cardiac-type KATP channels in intact cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Physiology and Membrane Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT

Background: 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 (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 findings: Single-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.

Conclusion: The 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.

Show MeSH
Related in: MedlinePlus