Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State

Overview

Journal bioRxiv

Date 2023 Jul 18

PMID 37461514

Authors

Bengt Svensson

Florentin R Nitu

Robyn T Rebbeck

Lindsey M McGurran

Tetsuro Oda

David D Thomas

Donald M Bers

Razvan L Cornea

Affiliations

Soon will be listed here.

Abstract

Ca leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide, (DPc10) detects leaky RyR2 in cardiomyocytes. Conversely, calmodulin (CaM) inhibits RyR2 leak. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. Here we used FRET to understand the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca , DPc10 decreased both FRET (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca , DPc10 decreased FRET without affecting CaM/RyR2 binding affinity. This correlates with analysis of fluorescence-lifetime-detected FRET indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca and lengthens D-FKBP/A-CaM distances independent of [Ca ]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, this effect being larger in micromolar vs. nanomolar Ca . Moreover, A-DPc10/RyR2 binding is cooperative in CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

References

Dhindwal S, Lobo J, Cabra V, Santiago D, Nayak A, Dryden K . A cryo-EM-based model of phosphorylation- and FKBP12.6-mediated allosterism of the cardiac ryanodine receptor. Sci Signal. 2017; 10(480). DOI: 10.1126/scisignal.aai8842. View

Dong X, Thomas D . Time-resolved FRET reveals the structural mechanism of SERCA-PLB regulation. Biochem Biophys Res Commun. 2014; 449(2):196-201. PMC: 4054823. DOI: 10.1016/j.bbrc.2014.04.166. View

Uchinoumi H, Yano M, Suetomi T, Ono M, Xu X, Tateishi H . Catecholaminergic polymorphic ventricular tachycardia is caused by mutation-linked defective conformational regulation of the ryanodine receptor. Circ Res. 2010; 106(8):1413-24. PMC: 2862146. DOI: 10.1161/CIRCRESAHA.109.209312. View

Balshaw D, Xu L, Yamaguchi N, Pasek D, Meissner G . Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor). J Biol Chem. 2001; 276(23):20144-53. DOI: 10.1074/jbc.M010771200. View

Yamamoto T, Ikemoto N . Peptide probe study of the critical regulatory domain of the cardiac ryanodine receptor. Biochem Biophys Res Commun. 2002; 291(4):1102-8. DOI: 10.1006/bbrc.2002.6569. View

Rebbeck R, Ginsburg K, Ko C, Fasoli A, Rusch K, Cai G . Synergistic FRET assays for drug discovery targeting RyR2 channels. J Mol Cell Cardiol. 2022; 168:13-23. PMC: 10088286. DOI: 10.1016/j.yjmcc.2022.04.002. View

Xu X, Yano M, Uchinoumi H, Hino A, Suetomi T, Ono M . Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction. Biochem Biophys Res Commun. 2010; 394(3):660-6. PMC: 2858291. DOI: 10.1016/j.bbrc.2010.03.046. View

Gong D, Chi X, Wei J, Zhou G, Huang G, Zhang L . Modulation of cardiac ryanodine receptor 2 by calmodulin. Nature. 2019; 572(7769):347-351. DOI: 10.1038/s41586-019-1377-y. View

Humphrey W, Dalke A, Schulten K . VMD: visual molecular dynamics. J Mol Graph. 1996; 14(1):33-8, 27-8. DOI: 10.1016/0263-7855(96)00018-5. View

10.

Rebbeck R, Nitu F, Rohde D, Most P, Bers D, Thomas D . S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex. J Biol Chem. 2016; 291(30):15896-907. PMC: 4957069. DOI: 10.1074/jbc.M115.713107. View

11.

Yamamoto T, Ikemoto N . Spectroscopic monitoring of local conformational changes during the intramolecular domain-domain interaction of the ryanodine receptor. Biochemistry. 2002; 41(5):1492-501. DOI: 10.1021/bi015581z. View

12.

Oda T, Yano M, Yamamoto T, Tokuhisa T, Okuda S, Doi M . Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure. Circulation. 2005; 111(25):3400-10. DOI: 10.1161/CIRCULATIONAHA.104.507921. View

13.

Fukuda M, Yamamoto T, Nishimura S, Kato T, Murakami W, Hino A . Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes. Biochem Biophys Res Commun. 2014; 448(1):1-7. DOI: 10.1016/j.bbrc.2014.03.152. View

14.

Sondergaard M, Tian X, Liu Y, Wang R, Chazin W, Chen S . Arrhythmogenic Calmodulin Mutations Affect the Activation and Termination of Cardiac Ryanodine Receptor-mediated Ca2+ Release. J Biol Chem. 2015; 290(43):26151-62. PMC: 4646266. DOI: 10.1074/jbc.M115.676627. View

15.

Yang Y, Guo T, Oda T, Chakraborty A, Chen L, Uchinoumi H . Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure. Circ Res. 2013; 114(2):295-306. PMC: 4004530. DOI: 10.1161/CIRCRESAHA.114.302857. View

16.

Oda T, Yang Y, Nitu F, Svensson B, Lu X, Fruen B . In cardiomyocytes, binding of unzipping peptide activates ryanodine receptor 2 and reciprocally inhibits calmodulin binding. Circ Res. 2012; 112(3):487-97. PMC: 3573756. DOI: 10.1161/CIRCRESAHA.111.300290. View

17.

Cornea R, Nitu F, Gruber S, Kohler K, Satzer M, Thomas D . FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel. Proc Natl Acad Sci U S A. 2009; 106(15):6128-33. PMC: 2662960. DOI: 10.1073/pnas.0813010106. View

18.

Ahern G, Junankar P, Dulhunty A . Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12. Biophys J. 1997; 72(1):146-62. PMC: 1184304. DOI: 10.1016/S0006-3495(97)78654-5. View

19.

Fruen B, Black D, Bloomquist R, Bardy J, Johnson J, Louis C . Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin. Biochemistry. 2003; 42(9):2740-7. DOI: 10.1021/bi0267689. View

20.

Laver D, Honen B, Lamb G, Ikemoto N . A domain peptide of the cardiac ryanodine receptor regulates channel sensitivity to luminal Ca2+ via cytoplasmic Ca2+ sites. Eur Biophys J. 2007; 37(4):455-67. DOI: 10.1007/s00249-007-0238-z. View