Mapping the Ryanodine Receptor FK506-binding Protein Subunit Using Fluorescence Resonance Energy Transfer*

The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca2+ release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [3H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model.

[1]  S. Huke,et al.  Kinetics of FKBP12.6 Binding to Ryanodine Receptors in Permeabilized Cardiac Myocytes and Effects on Ca Sparks , 2010, Circulation research.

[2]  F. van Petegem,et al.  Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations. , 2009, Structure.

[3]  M. Ikura,et al.  Crystal structure of type I ryanodine receptor amino-terminal β-trefoil domain reveals a disease-associated mutation “hot spot” loop , 2009, Proceedings of the National Academy of Sciences.

[4]  S. Zissimopoulos,et al.  FKBP12.6 binding of ryanodine receptors carrying mutations associated with arrhythmogenic cardiac disease. , 2009, The Biochemical journal.

[5]  David D. Thomas,et al.  FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel , 2009, Proceedings of the National Academy of Sciences.

[6]  I. Serysheva,et al.  Ryanodine Receptor Structure: Progress and Challenges* , 2009, Journal of Biological Chemistry.

[7]  S. Matecki,et al.  Hypernitrosylated ryanodine receptor/calcium release channels are leaky in dystrophic muscle , 2009, Nature Medicine.

[8]  M. Baker,et al.  Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel , 2008, Proceedings of the National Academy of Sciences.

[9]  H. Duff,et al.  Removal of FKBP12.6 Does Not Alter the Conductance and Activation of the Cardiac Ryanodine Receptor or the Susceptibility to Stress-induced Ventricular Arrhythmias* , 2007, Journal of Biological Chemistry.

[10]  Godfrey L. Smith,et al.  Overexpression of FK-506–Binding Protein 12.0 Modulates Excitation–Contraction Coupling in Adult Rabbit Ventricular Cardiomyocytes , 2007, Circulation research.

[11]  T. Wagenknecht,et al.  Three-Dimensional Localization of Serine 2808, a Phosphorylation Site in Cardiac Ryanodine Receptor* , 2007, Journal of Biological Chemistry.

[12]  S. Zissimopoulos,et al.  Redox Sensitivity of the Ryanodine Receptor Interaction with FK506-binding Protein* , 2007, Journal of Biological Chemistry.

[13]  S. Priori,et al.  Arrhythmogenesis in Catecholaminergic Polymorphic Ventricular Tachycardia: Insights From a RyR2 R4496C Knock-In Mouse Model , 2006 .

[14]  D. Bers Cardiac ryanodine receptor phosphorylation: target sites and functional consequences. , 2006, The Biochemical journal.

[15]  Paul D Allen,et al.  Structural characterization of the RyR1-FKBP12 interaction. , 2006, Journal of molecular biology.

[16]  S. Reiken,et al.  Analysis of calstabin2 (FKBP12.6)-ryanodine receptor interactions: rescue of heart failure by calstabin2 in mice. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[17]  刘金明,et al.  IL-13受体α2降低血吸虫病肉芽肿的炎症反应并延长宿主存活时间[英]/Mentink-Kane MM,Cheever AW,Thompson RW,et al//Proc Natl Acad Sci U S A , 2005 .

[18]  S. Hamilton,et al.  Effects of S-glutathionylation and S-nitrosylation on calmodulin binding to triads and FKBP12 binding to type 1 calcium release channels. , 2005, Antioxidants & redox signaling.

[19]  David D. Thomas,et al.  Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin. , 2005, Biochemistry.

[20]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[21]  D. Bers Macromolecular complexes regulating cardiac ryanodine receptor function. , 2004, Journal of molecular and cellular cardiology.

[22]  S. Reiken,et al.  Sudden Death in Familial Polymorphic Ventricular Tachycardia Associated With Calcium Release Channel (Ryanodine Receptor) Leak , 2004, Circulation.

[23]  D. H. Kim,et al.  N-terminal Region of FKBP12 Is Essential for Binding to the Skeletal Ryanodine Receptor* , 2004, Journal of Biological Chemistry.

[24]  S. Priori,et al.  FKBP12.6 Deficiency and Defective Calcium Release Channel (Ryanodine Receptor) Function Linked to Exercise-Induced Sudden Cardiac Death , 2003, Cell.

[25]  J. Johnson,et al.  Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin. , 2003, Biochemistry.

[26]  Montserrat Samsó,et al.  Apocalmodulin and Ca2+-Calmodulin Bind to Neighboring Locations on the Ryanodine Receptor* , 2002, The Journal of Biological Chemistry.

[27]  D. Burkhoff,et al.  PKA Phosphorylation Dissociates FKBP12.6 from the Calcium Release Channel (Ryanodine Receptor) Defective Regulation in Failing Hearts , 2000, Cell.

[28]  M. Carson,et al.  Structure of FKBP12.6 in complex with rapamycin. , 2000, Acta crystallographica. Section D, Biological crystallography.

[29]  S. Fleischer,et al.  Three Amino Acid Residues Determine Selective Binding of FK506-binding Protein 12.6 to the Cardiac Ryanodine Receptor* , 1999, The Journal of Biological Chemistry.

[30]  S. Fleischer,et al.  FK-binding Protein Is Associated with the Ryanodine Receptor of Skeletal Muscle in Vertebrate Animals* , 1998, The Journal of Biological Chemistry.

[31]  Michael Radermacher,et al.  Locations of Calmodulin and FK506-binding Protein on the Three-dimensional Architecture of the Skeletal Muscle Ryanodine Receptor* , 1997, The Journal of Biological Chemistry.

[32]  S. Fleischer,et al.  Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms. , 1997, The American journal of physiology.

[33]  A. Marks,et al.  Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein , 1994, Cell.

[34]  A. P. Timerman,et al.  The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor. , 1994, Cell calcium.

[35]  D. Livingston,et al.  PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism. , 1992, The Journal of biological chemistry.

[36]  N. Sigal,et al.  FKBP, the binding protein for the immunosuppressive drug, FK-506, is not an inhibitor of protein kinase C activity. , 1991, Biochemical and biophysical research communications.

[37]  N. Burres,et al.  Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin. , 1991, The Journal of biological chemistry.

[38]  T. Wagenknecht,et al.  Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor. , 2006, Biophysical journal.

[39]  R. Haugland The Handbook: A Guide to Fluorescent Probes and Labeling Technologies , 2005 .