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Enhancing calmodulin binding to ryanodine receptor is crucial to limit neuronal cell loss in Alzheimer disease.

ABSTRACT: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive neuronal cell loss. Recently, dysregulation of intracellular Ca²⁺ homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Here, we investigated (1) the pathogenic role of destabilization of ryanodine receptor (RyR2) in endoplasmic reticulum (ER) upon development of AD phenotypes in App^NL-G-F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic), and (2) the therapeutic effect of enhanced calmodulin (CaM) binding to RyR2. In the neuronal cells from App^NL-G-F mice, CaM dissociation from RyR2 was associated with AD-related phenotypes, i.e. Aβ accumulation, TAU phosphorylation, ER stress, neuronal cell loss, and cognitive dysfunction. Surprisingly, either genetic (by V3599K substitution in RyR2) or pharmacological (by dantrolene) enhancement of CaM binding to RyR2 reversed almost completely the aforementioned AD-related phenotypes, except for Aβ accumulation. Thus, destabilization of RyR2 due to CaM dissociation is most likely an early and fundamental pathogenic mechanism involved in the development of AD. The discovery that neuronal cell loss can be fully prevented simply by stabilizing RyR2 sheds new light on the treatment of AD.

SUBMITTER: Nakamura Y

PROVIDER: S-EPMC8012710 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Enhancing calmodulin binding to cardiac ryanodine receptor completely inhibits pressure-overload induced hypertrophic signaling

Project description:Purpose: Cardiac hypertrophy is a well-known major risk factor for poor prognosis in patients with cardiovascular diseases. Dysregulation of intracellular Ca2+ is involved in the pathogenesis of cardiac hypertrophy. However, the precise mechanism underlying cardiac hypertrophy remains elusive. Here, we investigated whether pressure-overload induced hypertrophy can be induced by destabilization of cardiac ryanodine receptor (RyR2) through calmodulin (CaM) dissociation and subsequent Ca2+ leakage, and whether it can be genetically rescued by enhancing the binding affinity of CaM to RyR2. Methods: To examine the role of CaM-RyR2 complex in the development of pressure-overload induced cardiac hypertrophy, whole transcriptome analysis using RNA-seq analysis in the hearts from WT and homozygous RyR2V3599K/V3599K (V3599K) mice with or without transverse aortic constriction (TAC) was performed to elucidate the RyR2 signaling pathway in the heart. Results: Gene expression increased in WT (+TAC) hearts compared to WT (-TAC) hearts; however, this increase was not observed in V3599K (+TAC) hearts. Conclusions: Enhanced CaM binding to RyR2 decreased expression of hypertrophy-related genes.

2020-10-07 | GSE158536 | GEO

Ca2+-dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains.

Project description:The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2?µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.

| S-EPMC6340113 | biostudies-literature

S100A1 and calmodulin compete for the same binding site on ryanodine receptor.

Project description:In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca(2+) release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca(2+)-S100A1 competes with Ca(2+)-calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca(2+)-S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616-3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca(2+)-S100A1. These same residues were shown previously to be important for RyR1 binding to Ca(2+)-CaM. A model for regulating muscle contraction is presented in which Ca(2+)-S100A1 and Ca(2+)-CaM compete directly for the same binding site on the ryanodine receptor.

| S-EPMC2546546 | biostudies-literature

Enhancing calmodulin binding to cardiac ryanodine receptor completely inhibits pressure-overload induced hypertrophic signaling

Project description:Enhancing calmodulin binding to cardiac ryanodine receptor completely inhibits pressure-overload induced hypertrophic signaling

| PRJNA665524 | ENA

In cardiomyocytes, binding of unzipping peptide activates ryanodine receptor 2 and reciprocally inhibits calmodulin binding.

Project description:One hypothesis for elevated Ca(2+) leak through cardiac ryanodine receptors (ryanodine receptor 2 [RyR2]) in heart failure is interdomain unzipping that can enhance aberrant channel activation. A peptide (domain peptide corresponding to RyR2 residues 2460-2495 [DPc10]) corresponding to RyR2 central domain residues 2460-2495 recapitulates this arrhythmogenic RyR2 leakiness by unzipping N-terminal and central domains. Calmodulin (CaM) and FK506-binding protein (FKBP12.6) bind to RyR2 and stabilize the closed channel. Little is known about DPc10 binding to the RyR2 and how that may interact with binding (and effects) of CaM and FKBP12.6 to RyR2.To measure, directly in cardiac myocytes, the kinetics and binding affinity of DPc10 to RyR2 and how that affects RyR2 interaction with FKBP12.6 and CaM.We used permeabilized rat ventricular myocytes and fluorescently labeled DPc10, FKBP12.6, and CaM. DPc10 access to its binding site is extremely slow in resting RyR2 but is accelerated by promoting RyR opening or unzipping (by unlabeled DPc10). RyR2-bound CaM (but not FKBP12.6) drastically slowed DPc10 binding. Conversely, DPc10 binding significantly reduced CaM (but not FKBP12.6) binding to the RyR2. Fluorescence resonance energy transfer measurements indicate that DPc10-binding and CaM-binding sites are separate and allow triangulation of the structural DPc10 binding locus on RyR2 vs FKBP12.6-binding and CaM-binding sites.DPc10-RyR2 binding is sterically limited by the resting zipped RyR2 state. CaM binding to RyR2 stabilizes this zipped state, whereas RyR2 activation or prebound DPc10 enhances DPc10 access. DPc10-binding and CaM-binding sites are distinct but are allosterically interacting RyR2 sites. Neither DPc10 nor FKBP12.6 influences RyR2 binding of the other.

| S-EPMC3573756 | biostudies-literature

S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex.

Project description:S100A1 has been suggested as a therapeutic agent to enhance myocyte Ca(2+) cycling in heart failure, but its molecular mode of action is poorly understood. Using FRET, we tested the hypothesis that S100A1 directly competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. Our FRET readout provides an index of acceptor-labeled CaM binding near donor-labeled FKBP (FK506-binding protein 12.6) on the cytoplasmic domain of RyR in isolated sarcoplasmic reticulum vesicles. S100A1 (0.01-400 μm) partially inhibited FRET (i.e. CaM binding), with Ki > 10 μm, for both RyR1 and RyR2. The high [S100A1] required for partial effects on FRET indicates a lack of competition by S100A1 on CaM/RyR binding under normal physiological conditions. High-resolution analysis of time-resolved FRET detects two structural states of RyR-bound CaM, which respond to [Ca(2+)] and are isoform-specific. The distribution of these structural states was perturbed only by high micromolar [S100A1], which promoted a shift of bound CaM to a lower FRET orientation (without altering the amount of CaM bound to RyR). Thus, high micromolar S100A1 does alter the CaM/RyR interaction, without involving competition. Nevertheless, submicromolar S100A1 can alter RyR function, an effect that is influenced by both [Ca(2+)] and [CaM]. We conclude that CaM and S100A1 can concurrently bind to and functionally modulate RyR1 and RyR2, but this does not involve direct competition at the RyR CaM binding site.

| S-EPMC4957069 | biostudies-literature

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts.

Project description:AimsThe channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure.Methods and resultsThe involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels.ConclusionReduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

| S-EPMC3584972 | biostudies-literature

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor.

Project description:A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+ These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction.

| S-EPMC5270481 | biostudies-literature

Calmodulin-binding locations on the skeletal and cardiac ryanodine receptors.

Project description:Ryanodine receptor types 1 (RyR1) and 2 (RyR2) are calcium release channels that are highly enriched in skeletal and cardiac muscle, respectively, where they play an essential role in excitation-contraction coupling. Apocalmodulin (apo-CaM) weakly activates RyR1 but inhibits RyR2, whereas Ca(2+)-calmodulin inhibits both isoforms. Previous cryo-EM studies showed distinctly different binding locations on RyR1 for the two states of CaM. However, recent studies employing FRET appear to challenge these findings. Here, using cryo-EM, we have determined that a CaM mutant that is incapable of binding calcium binds to RyR1 at the apo site, regardless of the calcium concentration. We have also re-determined the location of RyR1-bound Ca(2+)-CaM using uniform experimental conditions. Our results show the existence of the two overlapping but distinct binding sites for CaM in RyR1 and imply that the binding location switch is due to Ca(2+) binding to CaM, as opposed to direct effects of Ca(2+) on RyR1. We also discuss explanations that could resolve the apparent conflict between the cryo-EM and FRET results. Interestingly, apo-CaM binds to RyR2 at a similar binding location to that of Ca(2+)-CaM on RyR1, in seeming agreement with the inhibitory effects of these two forms of CaM on their respective receptors.

| S-EPMC3436284 | biostudies-literature

Nuclear translocation of calmodulin in pathological cardiac hypertrophy originates from ryanodine receptor bound calmodulin.

Project description:In cardiac myocytes Calmodulin (CaM) bound to the ryanodine receptor (RyR2) constitutes a large pool of total myocyte CaM, but the CaM-RyR2 affinity is reduced in pathological conditions. Knock-in mice expressing RyR2 unable to bind CaM also developed hypertrophy and early death. However, it is unknown whether CaM released from this RyR2-bound pool participates in pathological cardiac hypertrophy. We found that angiotensin II (AngII) or phenylephrine (PE) both cause CaM to dissociate from the RyR2 and translocate to the nucleus. To test whether this nuclear CaM accumulation depends on CaM released from RyR2, we enhanced CaM-RyR2 binding affinity (with dantrolene), or caused CaM dissociation from RyR2 (using suramin). Dantrolene dramatically reduced AngII- and PE-induced nuclear CaM accumulation. Conversely, suramin enhanced nuclear CaM accumulation. This is consistent with nuclear CaM accumulation coming largely from the CaM-RyR2 pool. CaM lacks a nuclear localization signal (NLS), but G-protein coupled receptor kinase 5 (GRK5) binds CaM, has a NLS and translocates like CaM in response to AngII or PE. Suramin also promoted GRK5 nuclear import, and caused nuclear export of histone deacetylase 5 (HDAC5). Dantrolene prevented these effects. After 2-8?weeks of pressure overload (TAC) CaM binding to RyR2 was reduced, nuclear CaM and GRK5 were both elevated and there was enhanced nuclear export of HDAC5. Stress (acute AngII or TAC) causes CaM dissociation from RyR2 and translocation to the nucleus with GRK5 with parallel HDAC5 nuclear export. Thus CaM dissociation from RyR2 may be an important step in driving pathological hypertrophic gene transcription.

| S-EPMC6402808 | biostudies-literature

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