iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://pubmed.ncbi.nlm.nih.gov/18755143
Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in rat cardiomyocytes - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Nov 7;376(1):80-5.
doi: 10.1016/j.bbrc.2008.08.084. Epub 2008 Aug 26.

Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in rat cardiomyocytes

Sabine Huke  1 , Donald M Bers
Affiliations

Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in rat cardiomyocytes

Sabine Huke et al. Biochem Biophys Res Commun. .

Abstract

The cardiac ryanodine receptor (RyR) controls Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling. Three phosphorylation sites have been identified: Serine-(S)2808, S2814 and recently S2030. We measured phosphorylation with at least two different antibodies per site and demonstrate that for S2808 results were highly antibody-dependent and two out of three S2808 antibodies did not accurately report phosphorylation level. The RyR was substantially phosphorylated in quiescent rat cardiomyocytes at S2808 and less so at S2814, but appeared to be unphosphorylated at S2030. Basal phosphorylation at S2808/S2814 was maintained by a Ca2+ dependent kinase other than Ca2+/Calmodulin-dependent kinase (CaMKII). During stimulation with Isoproterenol S2808 was phosphorylated by protein kinase A (PKA) and S2814 was phosphorylated by CaMKII. Phosphatase 1 appears to be the main phosphatase dephosphorylating S2808/S2814, but phosphatase 2a may also dephosphorylate S2814. RyR phosphorylation is complex, but important in understanding RyR functional modulation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Evaluation of phosphorylation-site specific antibodies using immunoprecipitated RyR treated with AP, alkaline phosphatase; PP1, phosphatase 1 or PKA. (A) Antigenic peptides for all three ANTI-PS2808 antibodies used. (B) The Marks ANTI-PS2808 antibody shows increased reactivity with the RyR after both, AP and PKA treatment. (C) All three ANTI-PS2808 antibodies detected a decrease in phosphorylation when samples were treated with PP1, but showed either a decrease (Colyer), increase (Marks) or no change (Chen) when samples were treated with AP. (D) The result is dependent on the duration of the AP treatment. This experiment and the one shown in (C) were repeated once more with a similar result.
Figure 2
Figure 2
(A, B) Summay of results (n=7–9) for changes in PLB ot RyR phosphorylation in cardiomyocytes stimulated with Iso (0.1 or 0.3 µM) and Iso + CalA. (C) Representative Western Blots (each panel is from the same gel, but intermediate lanes were deleted). (D) RyR phosphorylation at S2030 in intact cardiomyocytes in response to Iso measured with two different antibodies. The positive control (pos Ctr) sample serves as antibody control only and was not loaded proportionally.
Figure 3
Figure 3
(A) Effect of PKA/PKG inhibitor H89 (10–30 µM) on RyR phosphorylation with and without Iso. Number of independent experiments is indicated in last column. (B) Effect of PKA inhibitor PKI (10–30 µM). (C, D) Effect of CaMKII inhibition with KN93 (10 µM). Please note that the Iso treated cells in those experiments were electrically stimulated for 5 min at 2 Hz to increase CaMKII activation. (E) Representative Western Blot showing the effect of KN93 on RyR and PLB phosphorylation.
Figure 4
Figure 4
RyR dephosphorylation in vitro and in intact cells. (A) Immunoprecipitated RyR was treated with PP1 or PP2a for 10 and 30 min at 30°C. (B) Increase in phosphorylation at S2808 and S2814 in response to treatment with the phosphatase inhibitor okadaic acid (OA; n=3). The response to 1 µM CalA was set to 100.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J. Mol. Cell. Cardiol. 2004;37:417–429. - PubMed
    1. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000;101:365–376. - PubMed
    1. Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM. Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ. Res. 2005;97:1314–1322. - PubMed
    1. Wehrens XH, Lehnart SE, Marks AR. Ryanodine receptor-targeted anti-arrhythmic therapy. Ann. N. Y. Acad. Sci. 2005;1047:366–375. - PubMed
    1. Witcher DR, Kovacs RJ, Schulman H, Cefali DC, Jones LR. Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J. Biol. Chem. 1991;266:11144–11152. - PubMed

Publication types

MeSH terms