Peptide Probe Study of the Critical Regulatory Domain of the Cardiac Ryanodine Receptor

doi:10.1006/bbrc.2002.6569

Biochemical and Biophysical Research Communications

Volume 291, Issue 4, 8 March 2002, Pages 1102-1108

https://doi.org/10.1006/bbrc.2002.6569 Get rights and content

Abstract

The recently devised domain peptide probe technique was used to identify and characterize critical domains of the cardiac ryanodine receptor (RyR2). A synthetic peptide corresponding to the Gly²⁴⁶⁰-Pro²⁴⁹⁵ domain of the RyR2, designated DPc10, enhanced the ryanodine binding activity and increased the sensitivity of the RyR2 to activating Ca²⁺: the effects that resemble the typical phenotypes of cardiac diseases. A single Arg-to-Ser mutation made in DPc10, mimicking the recently reported Arg²⁴⁷⁴-to-Ser²⁴⁷⁴ human mutation, abolished all of these effects that would have been produced by DPc10. On the basis of the principle of the domain peptide probe approach (see Model 1), these results indicate that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. This domain peptide approach opens the new possibility in the studies of the regulatory and pathogenic mechanisms of the cardiac Ca²⁺ channel.

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Cited by (59)

Defective interactions of protein partner with ion channels and transporters as alternative mechanisms of membrane channelopathies
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The past twenty years have revealed the existence of numerous ion channel mutations resulting in human pathology. Ion channels provide the basis of diverse cellular functions, ranging from hormone secretion, excitation–contraction coupling, cell signaling, immune response, and trans-epithelial transport. Therefore, the regulation of biophysical properties of channels is vital in human physiology. Only within the last decade has the role of non-ion channel components come to light in regard to ion channel spatial, temporal, and biophysical regulation in physiology. A growing number of auxiliary components have been determined to play elemental roles in excitable cell physiology, with dysfunction resulting in disorders and related manifestations. This review focuses on the broad implications of such dysfunction, focusing on disease-causing mutations that alter interactions between ion channels and auxiliary ion channel components in a diverse set of human excitable cell disease. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé
FRET-based trilateration of probes bound within functional ryanodine receptors
2014, Biophysical Journal
Citation Excerpt :
Having validated our improved trilateration method, we then used it to determine the location of an acceptor probe attached to DPc10 bound to RyR2 based on FRET measurements carried out in permeabilized myocytes (Table 3). It has been postulated that DPc10 directly competes with intersubunit contacts (51) between the RyR2 N- and central domains (zipping) by binding to the N-domain sequence (to unzip) (18). Taken literally, this model implies that DPc10 should bind in close proximity to the N-domain zipping surface.
To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca²⁺ channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460–2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca²⁺ leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ∼35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.
Modeling a ryanodine receptor N-terminal domain connecting the central vestibule and the corner clamp region
2013, Journal of Biological Chemistry
Ryanodine receptors (RyRs) form a class of intracellular calcium release channels in various excitable tissues and cells such as muscles and neurons. They are the major cellular mediators of the release of calcium ions from the sarcoplasmic reticulum, an essential step in muscle excitation-contraction coupling. Several crystal structures of skeletal muscle RyR1 peptide fragments have been solved, but these cover less than 15% of the full-length RyR1 sequence. In this study, by combining modeling techniques with sub-nanometer resolution cryo-electron microscopy (cryo-EM) maps, we obtained pseudo-atomic models for RyR fragments consisting of residues 850–1,056 in rabbit RyR1 or residues 861–1,067 in mouse RyR2. These fragments are docked into a domain that connects the central vestibule and corner clamp region of RyR, resulting in a good match of the secondary structure elements in the cryo-EM map and the pseudo-atomic models, which is also consistent with our previous mappings of GFP insertions by cryo-EM and with FRET measurements involving RyR and FK506-binding protein (FKBP). A combined model of the RyR fragment and FKBP docked into the cryo-EM map suggests that the fragment is positioned adjacent to the FKBP-binding site. Its predicted binding interface with FKBP consists primarily of electrostatic contacts and contains several disease-associated mutations. A dynamic interaction between the fragment and an RyR phosphorylation domain, characterized by FRET experiments, also supports the structural predictions of the pseudo-atomic models.
Background: High resolution structural information only covers 15% of the full-length sequence of RyR.
Results: Pseudo-atomic structures are generated for RyR1 fragments 850–1,056 and RyR2 861–1,067, docked into cryo-EM maps, and supported by FRET experiments.
Conclusion: The binding interface between RyR and FKBP consists of electrostatic contacts and contains mutations.
Significance: The fragments docked into a domain that forms an intersubunit interaction with a phosphorylation domain.
Intracellular translocation of calmodulin and Ca<sup>2+</sup>/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes
2010, Biochemical and Biophysical Research Communications
We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca²⁺ leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 [3]). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca²⁺/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca²⁺ transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca²⁺ transients results in the appearance of trains of Ca²⁺ spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca²⁺ events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.
Ryanodine receptor mutations in arrhythmia: The continuing mystery of channel dysfunction
2010, FEBS Letters
Mutations in RyR2 are causative of an inherited disorder which often results in sudden cardiac death. Dysfunctional channel behaviour has been the subject of many investigations varying from single channel analysis through to complex animal models. This review discusses recent advances in the field, describes the controversy surrounding the exact consequences of RyR2 mutation and how the disparate data may be reconciled. This heterogeneity of function with respect to the effects of polymorphisms, phosphorylation, cytosolic and luminal Ca²⁺ as well as inter-domain interactions may have important implications for the recent pharmaceutical therapies which have been put forward. We surmise that a comprehensive characterisation of mutations on a case-by-case basis may be beneficial for the development of specifically targeted therapies.
Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2
2010, Protein Expression and Purification
We report the domain analysis of the N-terminal region (residues 1–759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR2^1–606·His₆, RyR2^391–606·His₆, RyR2^409–606·His₆, Trx·RyR2^384–606·His₆, Trx·RyR2^391-606·His₆ and Trx·RyR2^409–606·His₆. The folding of RyR2^1–606·His₆ was analyzed by circular dichroism spectroscopy resulting in α-helix and β-sheet content of ∼23% and ∼29%, respectively, at temperatures up to 35 °C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR2^1–606·His₆, resulted in the appearance of two specific subfragments of ∼40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His₆·Tag antibody indicated that RyR2^1–606·His₆ is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively.

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^☆: Abbreviations used: RyR, ryanodine receptor; MH, malignant hyperthermia; CCD, central core disease; PMSF, phenylmethanesulfonyl fluoride; MES, 2-(N-morpholino) ethanesulfonic acid; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; MOPS, 3-(N-morpholino) propanesulfonic acid; VT, ventricular tachycardia; ARVD, arrhythmogenic right ventricular dysplasia.

^☆☆: This work was supported by National Institutes of Health Grant AR 16922.

²: To whom correspondence should be addressed at Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Fax: 617-972-1761. E-mail: [email protected].

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Regular ArticlePeptide Probe Study of the Critical Regulatory Domain of the Cardiac Ryanodine Receptor☆,☆☆

Abstract

Am. J. Med.

J. Biol. Chem.

J. Biol. Chem.

Trends. Cardiovasc. Med.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Ryanodine receptor mutations in malignant hyperthermia and central core disease

Hum. Mutat.

Regular Article
Peptide Probe Study of the Critical Regulatory Domain of the Cardiac Ryanodine Receptor☆,☆☆