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

2019 ◽  
Vol 476 (2) ◽  
pp. 193-209 ◽  
Author(s):  
Malene Brohus ◽  
Mads T. Søndergaard ◽  
Sui Rong Wayne Chen ◽  
Filip van Petegem ◽  
Michael T. Overgaard
Keyword(s):  
Ryanodine Receptor ◽  
Fluorescence Anisotropy ◽  
Dependent Manner ◽  
Binding Model ◽  
Calmodulin Binding ◽  
Binding Domains ◽  
Wide Range ◽  
Dependent Inhibition ◽  
Life Threatening ◽  
Cardiac Ryanodine Receptor

Abstract 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.

Role of cardiac ryanodine receptor calmodulin‐binding domains in mediating the action of arrhythmogenic calmodulin N‐domain mutation N54I

FEBS Journal ◽  
2019 ◽  
Vol 287 (11) ◽  
pp. 2256-2280 ◽  
Author(s):  
Mads T. Søndergaard ◽  
Yingjie Liu ◽  
Wenting Guo ◽  
Jinhong Wei ◽  
Ruiwu Wang ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Calmodulin Binding ◽  
Binding Domains ◽  
Cardiac Ryanodine Receptor

scholarly journals Short fungal fractions of β-1,3 glucans affect platelet activation

2016 ◽  
Vol 311 (3) ◽  
pp. H725-H734 ◽  
Author(s):  
Hélène Vancraeyneste ◽  
Rogatien Charlet ◽  
Yann Guerardel ◽  
Laura Choteau ◽  
Anne Bauters ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Atp Release ◽  
Human Platelets ◽  
Receptor Expression ◽  
Dependent Manner ◽  
Platelet Activity ◽  
Concentration Dependent Manner ◽  
Wide Range ◽  
Invasive Infections ◽  
Life Threatening

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains β-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of β-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans. The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 μmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbβ3. BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans. GLc5 decreased ATP release and TGF-β1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.

scholarly journals The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor

2020 ◽  
Vol 295 (22) ◽  
pp. 7620-7634
Author(s):  
Christian Holt ◽  
Louise Hamborg ◽  
Kelvin Lau ◽  
Malene Brohus ◽  
Anders Bundgaard Sørensen ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Ryanodine Receptor ◽  
Hydrophobic Surface ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Open Probability ◽  
Calmodulin Binding ◽  
Terminal Domain ◽  
Genes Encoding ◽  
Cardiac Ryanodine Receptor

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia–causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84–2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.

Correction of Defective Calmodulin Binding to the Cardiac Ryanodine Receptor Inhibits Aberrant Ca2+ Release in CPVT-associated Channel Dysfunction

2012 ◽  
Vol 18 (10) ◽  
pp. S188
Author(s):  
Masakazu Fukuda ◽  
Masafumi Yano ◽  
Takayoshi Kato ◽  
Hiroki Tateishi ◽  
Masahiro Doi ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Calmodulin Binding ◽  
Cardiac Ryanodine Receptor

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

2016 ◽  
Vol 292 (4) ◽  
pp. 1385-1395 ◽  
Author(s):  
Mads Toft Søndergaard ◽  
Yingjie Liu ◽  
Kamilla Taunsig Larsen ◽  
Alma Nani ◽  
Xixi Tian ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Dependent Inhibition ◽  
Cardiac Ryanodine Receptor

Enhancement of Calmodulin Binding to Cardiac Ryanodine Receptor Corrects the Defective Channel Gating in Failing Hearts

2011 ◽  
Vol 17 (9) ◽  
pp. S170
Author(s):  
Akihiro Hino ◽  
Masafumi Yano ◽  
Takayoshi Katoh ◽  
Masakazu Fukuda ◽  
Takeshi Suetomi ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Channel Gating ◽  
Calmodulin Binding ◽  
Cardiac Ryanodine Receptor

Interaction of the Lys3614–Asn3643 calmodulin-binding domain with the Cys4114-Asn4142 region of the type 1 ryanodine receptor is involved in the mechanism of Ca2+/agonist-induced channel activation

2008 ◽  
Vol 411 (2) ◽  
pp. 415-423 ◽  
Author(s):  
Jaya P. Gangopadhyay ◽  
Noriaki Ikemoto
Keyword(s):  
Ryanodine Receptor ◽  
Binding Activity ◽  
Significant Inhibition ◽  
Binding Domain ◽  
Domain Pair ◽  
Dependent Manner ◽  
Domain Interaction ◽  
Channel Activation ◽  
Calmodulin Binding

In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys3614–Asn3643) with the Cys4114–Asn4142 region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys3614–Asn3643) and anti-(Cys4114–Asn4142) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys3614–Asn3643 and Cys4114–Asn4142, activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a ‘channel-activation link’ between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03–2.0 μM, as determined by Stern–Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 μM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys3614–Asn3643 and Cys4114–Asn4142 regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.

scholarly journals Conserved properties of individual Ca2+-binding sites in calmodulin

2016 ◽  
Vol 113 (9) ◽  
pp. E1216-E1225 ◽  
Author(s):  
D. Brent Halling ◽  
Benjamin J. Liebeskind ◽  
Amelia W. Hall ◽  
Richard W. Aldrich
Keyword(s):  
Binding Sites ◽  
Evolutionary Analysis ◽  
Dependent Manner ◽  
Target Proteins ◽  
Wide Range ◽  
Ef Hand ◽  
Life Threatening ◽  
Ef Hands ◽  
Future Work ◽  
The Relationship

Calmodulin (CaM) is a Ca2+-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca2+ concentration into cellular signals by binding to a wide range of target proteins in a Ca2+-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca2+-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca2+ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.

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