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J. Biol. Chem., Vol. 282, Issue 35, 25929-25939, August 31, 2007

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Three-Dimensional Localization of Serine 2808, a Phosphorylation Site in Cardiac Ryanodine Receptor^*

Xing Meng, Bailong Xiao, Shitian Cai, Xiaojun Huang^¶, Fei Li^¶, Jeff Bolstad, Ramon Trujillo, Judith Airey^||, S. R. Wayne Chen, Terence Wagenknecht^¶, and Zheng Liu¹

From the Wadsworth Center, New York State Department of Health, Albany, New York 12201, the Departments of Physiology and Biophysics, and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the ^¶Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, New York 12201, and the ^||Department of Pharmacology, University of Nevada, Reno, Nevada 89557

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Phosphorylation of RyR2 by cAMP-dependent protein kinase A and by calmodulin-dependent protein kinase II modulates channel activity. Hyperphosphorylation at a single amino acid residue, Ser-2808, has been proposed to directly disrupt the binding of a 12.6-kDa FK506-binding protein (FKBP12.6) to RyR2, causing a RyR2 malfunction that triggers cardiac arrhythmias in human heart failure. To determine the structural basis of the interaction between Ser-2808 and FKBP12.6, we have employed two independent approaches to map this phosphorylation site in RyR2 by three-dimensional cryo-electron microscopy. In one approach, we inserted a green fluorescent protein (GFP) after amino acid Tyr-2801, and mapped the GFP three-dimensional location in the RyR2 structure. In another approach, the binding site of monoclonal antibody 34C was mapped in the three-dimensional structure of skeletal muscle RyR1. The epitope of antibody 34C has been mapped to amino acid residues 2,756 through 2,803 of the RyR1 sequence, corresponding to residues 2,722 through 2,769 of the RyR2 sequence. These locations of GFP insertion and antibody binding are adjacent to one another in domain 6 of the cytoplasmic clamp region. Importantly, the three-dimensional location of the Ser-2808 phosphorylation site is 105-120Å distance from the FKBP12.6 binding site mapped previously, indicating that Ser-2808 is unlikely to be directly involved in the binding of FKBP12.6 to RyR2, as had been proposed previously.

Received for publication, May 31, 2007

^* This work was supported in part by American Heart Association Grant 0430076N (to Z. L.), by National Institutes of Health Grant AR40615 (to T. W.), and by research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta (to S. R. W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Wadsworth Center, NY State Dept. of Health, Albany, NY 12201. Tel.: 518-474-6516; Fax: 518-474-7992; E-mail: liuz{at}wadsworthorg.

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