PKA (protein kinase A)-dependent phosphorylation of the cardiac Ca2+-release channel/RyR2 (type 2 ryanodine receptor) is believed to directly dissociate FKBP12.6 (12.6 kDa FK506-binding protein) from the channel, causing abnormal channel activation and Ca2+ release. To gain insight into the structural basis of the regulation of RyR2 by PKA, we determined the three-dimensional location of the PKA site Ser2030. GFP (green fluorescent protein) was inserted into RyR2-wt (wild-type RyR2) and RyR2 mutant, A4860G, after Thr2023. The resultant GFP–RyR2 fusion proteins, RyR2T2023-GFP and RyR2(A4860G)T2023-GFP, were expressed in HEK-293 (human embryonic kidney) cells and functionally characterized. Ca2+-release assays revealed that both GFP–RyR2 fusion proteins formed caffeine- and ryanodine-sensitive Ca2+-release channels. Further analyses using [3H]ryanodine binding demonstrated that the insertion of GFP into RyR2-wt after Thr2023 reduced the sensitivity of the channel to activation by Ca2+ or caffeine. RyR2(A4860G)T2023-GFP was found to be structurally more stable than RyR2T2023-GFP and was subsequently used as a basis for three-dimensional reconstruction. Cryo-electron microscopy and single particle image processing of the purified RyR2(A4860G)T2023-GFP protein revealed the location of the inserted GFP, and hence the Ser2030 PKA site in domain 4, a region that may be involved in signal transduction between the transmembrane and cytoplasmic domains. Like the Ser2808 PKA site reported previously, the Ser2030 site is not located close to the FKBP12.6-binding site mapped previously, indicating that neither of these PKA sites is directly involved in FKBP12.6 binding. On the basis of the three-dimensional localizations of a number of residues or regions, a model for the subunit organization in the structure of RyR2 is proposed.
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March 2008
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February 12 2008
Localization of PKA phosphorylation site, Ser2030, in the three-dimensional structure of cardiac ryanodine receptor Available to Purchase
Peter P. Jones;
Peter P. Jones
*Department of Physiology and Biophysics, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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Xing Meng;
Xing Meng
†Wadsworth Center, New York State Department of Health, Albany, NY 12201, U.S.A.
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Bailong Xiao;
Bailong Xiao
*Department of Physiology and Biophysics, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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Shitian Cai;
Shitian Cai
*Department of Physiology and Biophysics, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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Jeff Bolstad;
Jeff Bolstad
*Department of Physiology and Biophysics, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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Terence Wagenknecht;
Terence Wagenknecht
†Wadsworth Center, New York State Department of Health, Albany, NY 12201, U.S.A.
‡Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12201, U.S.A.
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Zheng Liu;
Zheng Liu
1
†Wadsworth Center, New York State Department of Health, Albany, NY 12201, U.S.A.
1Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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S. R. Wayne Chen
S. R. Wayne Chen
1
*Department of Physiology and Biophysics, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada T2N 4N1
1Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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Publisher: Portland Press Ltd
Received:
September 12 2007
Revision Received:
October 11 2007
Accepted:
October 30 2007
Accepted Manuscript online:
October 30 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 410 (2): 261–270.
Article history
Received:
September 12 2007
Revision Received:
October 11 2007
Accepted:
October 30 2007
Accepted Manuscript online:
October 30 2007
Citation
Peter P. Jones, Xing Meng, Bailong Xiao, Shitian Cai, Jeff Bolstad, Terence Wagenknecht, Zheng Liu, S. R. Wayne Chen; Localization of PKA phosphorylation site, Ser2030, in the three-dimensional structure of cardiac ryanodine receptor. Biochem J 1 March 2008; 410 (2): 261–270. doi: https://doi.org/10.1042/BJ20071257
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