The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Nicole A. Beard - One of the best experts on this subject based on the ideXlab platform.
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Ca^2+ signaling in striated muscle: the elusive roles of triadin, junctin, and Calsequestrin
European Biophysics Journal, 2009Co-Authors: Nicole A. Beard, Angela Fay DulhuntyAbstract:This review focuses on molecular interactions between Calsequestrin, triadin, junctin and the ryanodine receptor in the lumen of the sarcoplasmic reticulum. These interactions modulate changes in Ca^2+ release in response to changes in the Ca^2+ load within the sarcoplasmic reticulum store in striated muscle and are of fundamental importance to Ca^2+ homeostasis, since massive adaptive changes occur when expression of the proteins is manipulated, while mutations in Calsequestrin lead to functional changes which can be fatal. We find that Calsequestrin plays a different role in the heart and skeletal muscle, enhancing Ca^2+ release in the heart, but depressing Ca^2+ release in skeletal muscle. We also find that triadin and junctin exert independent influences on the ryanodine receptor in skeletal muscle where triadin alone modifies excitation–contraction coupling, while junctin alone supports functional interactions between Calsequestrin and the ryanodine receptor.
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Ca2+ signaling in striated muscle: the elusive roles of triadin, junctin, and Calsequestrin
European biophysics journal : EBJ, 2009Co-Authors: Nicole A. Beard, Lan Wei, Angela F. DulhuntyAbstract:This review focuses on molecular interactions between Calsequestrin, triadin, junctin and the ryanodine receptor in the lumen of the sarcoplasmic reticulum. These interactions modulate changes in Ca2+ release in response to changes in the Ca2+ load within the sarcoplasmic reticulum store in striated muscle and are of fundamental importance to Ca2+ homeostasis, since massive adaptive changes occur when expression of the proteins is manipulated, while mutations in Calsequestrin lead to functional changes which can be fatal. We find that Calsequestrin plays a different role in the heart and skeletal muscle, enhancing Ca2+ release in the heart, but depressing Ca2+ release in skeletal muscle. We also find that triadin and junctin exert independent influences on the ryanodine receptor in skeletal muscle where triadin alone modifies excitation–contraction coupling, while junctin alone supports functional interactions between Calsequestrin and the ryanodine receptor.
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Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with Calsequestrin
The international journal of biochemistry & cell biology, 2009Co-Authors: Lan Wei, Angela F. Dulhunty, Esther M. Gallant, Nicole A. BeardAbstract:Abstract Normal Ca2+ signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca2+ binding protein Calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca2+ stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle Calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal Calsequestrin and ryanodine receptors. Purified Calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the Calsequestrin re-associated channel complexes were further inhibited when luminal Ca2+ fell from 1 mM to ≤100 μM, as seen with native channels (containing endogenous Calsequestrin/triadin/junctin). In contrast, skeletal Calsequestrin had no effect on the triadin/ryanodine receptor complex and the channel activity of this complex increased when luminal Ca2+ fell, as seen with purified channels prior to triadin/Calsequestrin re-association. Therefore in this cell free system, junctin alone mediates signals between luminal Ca2+, skeletal Calsequestrin and skeletal ryanodine receptors and may curtail resting Ca2+ leak from the sarcoplasmic reticulum. We suggest that triadin serves a different function which may dominate during excitation–contraction coupling.
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Phosphorylation of skeletal muscle Calsequestrin enhances its Ca2+ binding capacity and promotes its association with junctin.
Cell calcium, 2008Co-Authors: Nicole A. Beard, Magdolna Varsányi, Lan Wei, Stephanie N. Cheung, Takashi Kimura, Angela F. DulhuntyAbstract:Summary Calcium signaling, intrinsic to skeletal and cardiac muscle function, is critically dependent on the amount of calcium stored within the sarcoplasmic reticulum. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the ryanodine receptor and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. We examined the influence of phosphorylation of Calsequestrin on its ability to store calcium, to polymerise and to regulate ryanodine receptors by binding to triadin and junctin. Our hypothesis was that these parameters might be altered by phosphorylation of threonine 353, which is located near the calcium and triadin/junctin binding sites. Although phosphorylation increased the calcium binding capacity of Calsequestrin nearly 2-fold, it did not alter Calsequestrin polymerisation, its binding to triadin or junctin or inhibition of ryanodine receptor activity at 1 mM luminal calcium. Phosphorylation was required for Calsequestrin binding to junctin when calcium concentration was low (100 nM), and ryanodine receptors were activated by dephosphorylated Calsequestrin when it bound to triadin alone. These novel data shows that phosphorylated Calsequestrin is required for high capacity calcium buffering and suggest that ryanodine receptor inhibition by Calsequestrin is mediated by junctin.
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antibodies targeting the calcium binding skeletal muscle protein Calsequestrin are specific markers of ophthalmopathy and sensitive indicators of ocular myopathy in patients with graves disease
Clinical and Experimental Immunology, 2006Co-Authors: Bamini Gopinath, Nicole A. Beard, Cherielee Adams, Reilly Musselman, Junichi Tani, S Elkaissi, Jack R WallAbstract:We have identified several eye muscle antigens and studied the significance of the corresponding serum autoantibodies in patients with Graves' disease. Of these antigens, only Calsequestrin is expressed more in eye muscle than other skeletal muscles, which could explain at least partly the specific involvement of eye muscle in patients with Graves' disease. Earlier, we found a modest relationship between anti-Calsequestrin antibodies and ophthalmopathy, but in that study we used Calsequestrin prepared from rabbit heart muscle and measured antibodies by immunoblotting. We have reinvestigated the prevalences of anti-Calsequestrin antibodies in larger groups of well-characterized patients with thyroid autoimmunity with and without ophthalmopathy and control patients and healthy subjects, using standard enzyme-linked immunosorbent assay incorporating highly purified rabbit skeletal muscle Calsequestrin, which has a 97% homology with human Calsequestrin, as antigen. Anti-Calsequestrin antibodies were detected in 78% of patients with active congestive ophthalmopathy, in 92% of those with active inflammation and eye muscle involvement, but in only 22% of patients with chronic, 'burnt out' disease. Tests were also positive in 5% of patients with Graves' hyperthyroidism without evident ophthalmopathy (two patients) and one patient with 'watery eyes' but no other clear signs of congestive ophthalmopathy and IgA nephropathy and no known thyroid disease, but in no patient with Hashimoto's thyroiditis, toxic nodular goitre, non-toxic multi-nodular goitre or diabetes, or age- and sex-matched healthy subjects. In serial studies of all 11 patients with Graves' hyperthyroidism who had active ophthalmopathy at the time of the first clinic visit, or developed eye signs during the first 6 months, and positive anti-Calsequestrin antibodies in at least one sample, anti-Calsequestrin antibodies correlated with the onset of ocular myopathy in six patients. Antibodies targeting Calsequestrin appear to be specific markers for ophthalmopathy and sensitive indicators of the ocular myopathy subtype of ophthalmopathy in patients with thyroid autoimmunity. However, these results must be considered preliminary until a large prospective study of patients with newly diagnosed Graves' hyperthyroidism, in which serum levels of Calsequestrin antibodies are correlated with clinical changes and orbital eye muscle and connective tissue/fat volumes, has been carried out.
Angela F. Dulhunty - One of the best experts on this subject based on the ideXlab platform.
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Ca2+ signaling in striated muscle: the elusive roles of triadin, junctin, and Calsequestrin
European biophysics journal : EBJ, 2009Co-Authors: Nicole A. Beard, Lan Wei, Angela F. DulhuntyAbstract:This review focuses on molecular interactions between Calsequestrin, triadin, junctin and the ryanodine receptor in the lumen of the sarcoplasmic reticulum. These interactions modulate changes in Ca2+ release in response to changes in the Ca2+ load within the sarcoplasmic reticulum store in striated muscle and are of fundamental importance to Ca2+ homeostasis, since massive adaptive changes occur when expression of the proteins is manipulated, while mutations in Calsequestrin lead to functional changes which can be fatal. We find that Calsequestrin plays a different role in the heart and skeletal muscle, enhancing Ca2+ release in the heart, but depressing Ca2+ release in skeletal muscle. We also find that triadin and junctin exert independent influences on the ryanodine receptor in skeletal muscle where triadin alone modifies excitation–contraction coupling, while junctin alone supports functional interactions between Calsequestrin and the ryanodine receptor.
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Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with Calsequestrin
The international journal of biochemistry & cell biology, 2009Co-Authors: Lan Wei, Angela F. Dulhunty, Esther M. Gallant, Nicole A. BeardAbstract:Abstract Normal Ca2+ signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca2+ binding protein Calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca2+ stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle Calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal Calsequestrin and ryanodine receptors. Purified Calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the Calsequestrin re-associated channel complexes were further inhibited when luminal Ca2+ fell from 1 mM to ≤100 μM, as seen with native channels (containing endogenous Calsequestrin/triadin/junctin). In contrast, skeletal Calsequestrin had no effect on the triadin/ryanodine receptor complex and the channel activity of this complex increased when luminal Ca2+ fell, as seen with purified channels prior to triadin/Calsequestrin re-association. Therefore in this cell free system, junctin alone mediates signals between luminal Ca2+, skeletal Calsequestrin and skeletal ryanodine receptors and may curtail resting Ca2+ leak from the sarcoplasmic reticulum. We suggest that triadin serves a different function which may dominate during excitation–contraction coupling.
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Phosphorylation of skeletal muscle Calsequestrin enhances its Ca2+ binding capacity and promotes its association with junctin.
Cell calcium, 2008Co-Authors: Nicole A. Beard, Magdolna Varsányi, Lan Wei, Stephanie N. Cheung, Takashi Kimura, Angela F. DulhuntyAbstract:Summary Calcium signaling, intrinsic to skeletal and cardiac muscle function, is critically dependent on the amount of calcium stored within the sarcoplasmic reticulum. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the ryanodine receptor and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. We examined the influence of phosphorylation of Calsequestrin on its ability to store calcium, to polymerise and to regulate ryanodine receptors by binding to triadin and junctin. Our hypothesis was that these parameters might be altered by phosphorylation of threonine 353, which is located near the calcium and triadin/junctin binding sites. Although phosphorylation increased the calcium binding capacity of Calsequestrin nearly 2-fold, it did not alter Calsequestrin polymerisation, its binding to triadin or junctin or inhibition of ryanodine receptor activity at 1 mM luminal calcium. Phosphorylation was required for Calsequestrin binding to junctin when calcium concentration was low (100 nM), and ryanodine receptors were activated by dephosphorylated Calsequestrin when it bound to triadin alone. These novel data shows that phosphorylated Calsequestrin is required for high capacity calcium buffering and suggest that ryanodine receptor inhibition by Calsequestrin is mediated by junctin.
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The conformation of Calsequestrin determines its ability to regulate skeletal ryanodine receptors.
Biophysical Journal, 2006Co-Authors: Magdolna Varsányi, Angela F. Dulhunty, Nicole A. BeardAbstract:Ca2+ efflux from the sarcoplasmic reticulum decreases when store Ca2+ concentration falls, particularly in skinned fibers and isolated vesicles where luminal Ca2+ can be reduced to very low levels. However ryanodine receptor activity in many single channel studies is higher when the luminal free Ca2+ concentration is reduced. We investigated the hypothesis that prolonged exposure to low luminal Ca2+ causes conformational changes in Calsequestrin and deregulation of ryanodine receptors, allowing channel activity to increase. Lowering of luminal Ca2+ from 1 mM to 100 μM for several minutes resulted in conformational changes with dissociation of 65–75% of Calsequestrin from the junctional face membrane. The Calsequestrin remaining associated no longer regulated channels. In the absence of this regulation, ryanodine receptors were more active when luminal Ca2+ was lowered from 1 mM to 100 μM. In contrast, when ryanodine receptors were Calsequestrin regulated, lowering luminal Ca2+ either did not alter or decreased activity. Ryanodine receptors are regulated by Calsequestrin under physiological conditions where Calsequestrin is polymerized. Since depolymerization occurs slowly, Calsequestrin can regulate the ryanodine receptor and prevent excess Ca2+ release when the store is transiently depleted, for example, during high frequency activity or early stages of muscle fatigue.
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regulation of ryanodine receptors by Calsequestrin effect of high luminal ca2 and phosphorylation
Biophysical Journal, 2005Co-Authors: Nicole A. Beard, Magdolna Varsányi, Derek R Laver, Marco G Casarotto, Angela F. DulhuntyAbstract:Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that Calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which Calsequestrin dissociates from the ryanodine receptor and the effect of Calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of ≥4 mM dissociates Calsequestrin from junctional face membrane, whereas in the range of 1–3 mM Calsequestrin remains attached; 2), the association with Calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of Calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that Calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.
Eduardo Ríos - One of the best experts on this subject based on the ideXlab platform.
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Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Carlo Manno, Chulhee Kang, Lourdes Figueroa, Dirk Gillespie, Robert H Fitts, Clara Franziniarmstrong, Eduardo RíosAbstract:Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does Calsequestrin do it? To bind large amounts of calcium in vitro, Calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged Calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of Calsequestrin as [Ca2+] in the SR--measured with a Calsequestrin-fused biosensor--was lowered. We found that Calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca2+] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of Calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of Calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by Calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric Calsequestrin network and catastrophic structural changes in the SR.
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Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle.
International journal of molecular sciences, 2016Co-Authors: Kevin M. Lewis, Gerhard R. Munske, Samuel S. Byrd, Jeehoon Kang, Hyun Jai Cho, Eduardo Ríos, Chulhee KangAbstract:Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian Calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle Calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle Calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal Calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc2Man1), while cardiac Calsequestrin had five additional mannoses (GlcNAc2Man6). Skeletal Calsequestrin was not phosphorylated, while the C-terminal tails of cardiac Calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac Calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca2+-dependent polymerization capabilities of native bovine skeletal Calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic “guiderail”-like scaffold for Calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca2+ concentrations, and proper glycosylation, in turn, guarantees both effective Ca2+ storage/buffering of the sarcoplasmic reticulum and localization of Calsequestrin (Casq) at its target site.
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imaging studies of Calsequestrin structure in skeletal muscle effects of calcium release
Biophysical Journal, 2016Co-Authors: Eduardo Ríos, Carlo Manno, Lourdes Figueroa, Clara FranziniarmstrongAbstract:Studying Ca2+ release flux and FRAP of fluorescently tagged proteins, Manno et al (this meeting) showed that SR Ca2+ depletion results in impairment of Ca2+ signaling and disassembly of the Calsequestrin polymer that fills SR terminal cisternae (TC) in resting myofibers. We sought evidence of depolymerization in images of fluorescence of D4cpV-Calsequestrin, a FRET biosensor that targets TC and measures [Ca2+]SR. Vertical (z-) stacks of fluorescence images allowed for identification of subcellular structures (resolution was ca. 0.3 μm in xy and 0.6 μm in z, after deblurring). Depletion was induced by solutions with high K+, 0.5-5 mM chloro-M-cresol (CMC), plus pump inhibitors, and was verified by FRET of D4cpV. Upon depletion, the fluorescence transitioned from a pattern limited to TC to a more diffuse one, consistent with mobilization of Calsequestrin away from TC. Fourier analysis of these images showed migration of power away from the fundamental and into the lower harmonics of the power spectrum. Muscles depleted by solutions with 5 mM CMC, imaged by electron microscopy, showed electron-dense content —Calsequestrin-- in longitudinal sacs that normally appear empty. These images also showed major changes in membrane structure, which raise the possibility that the high CMC resulted in irreversible changes. After SR depletion by fatiguing tetanic stimulation of intact muscles the fluorescence of D4cpV-Calsequestrin redistributed to a lesser extent. This first demonstration of Calsequestrin depolymerization in vivo introduces the loss of Ca2+ buffering associated with this change as a previously unrecognized mechanism for functional impairment in muscle fatigue, suggests ways in which Calsequestrin could mediate gating control of RyR channels by [Ca2+]SR and justifies disease phenotypes linked to mutations of Calsequestrin that prevent its correct polymerization (see poster by Lewis et al, this meeting). Supported by NIGMS/NIH grant GM111254.
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Calsequestrin Depolymerizes when Ca2+ Concentration Decays in the Sarcoplasmic Reticulum of Skeletal Muscle
Biophysical Journal, 2016Co-Authors: Carlo Manno, Lourdes Figueroa, Dirk Gillespie, Eduardo RíosAbstract:Calsequestrin is the major Ca2+ binding protein of the sarcoplasmic reticulum (SR). In solution, it polymerizes as [Ca2+] increases, which in turn endows the protein with greater Ca2+ binding capacity. Within resting muscles Calsequestrin appears as linear polymers that fill SR terminal cisternae, but there is no evidence that it depolymerizes when Ca2+ leaves the SR. In voltage-clamped mouse myofibers depolarization-induced Ca2+ release flux features an early peak, followed by a “hump” of Ca2+ stored in Calsequestrin (Royer et al. JGP 2010). We measured the effects of prior Ca2+ release on release kinetics, using a conditioning-test pulse protocol. At the shortest intervals after the conditioning release, T=100 ms, peak flux was reduced by 70% and the hump was absent. As T increased the peak recovered with τ=207 ms, but the hump required T>30s for recovery. [Ca2+]SR recovered with τ=240 ms (SEM 53 ms, 11 cells). Thus, restoration of peak flux tracks recovery of the driving [Ca2+] gradient, but restoring the hump takes much longer. It follows that upon releasing Ca2+, Calsequestrin enters a long-lasting state that cancels its ability to support Ca2+ release. The hypothesis that the change consists in depolymerization was tested by FRAP of EYFP-Calsequestrin and D4cpV-Calsequestrin, which revealed an inverse relationship between Calsequestrin's diffusion coefficient (Deff) and [Ca2+]SR, consistent with Calsequestrin's depolymerization upon reduction of [Ca2+]SR. A similar dependence was found for the Deff of [Ca2+]SR monitor D1ER, indicating that the polymeric network hinders diffusion of species other than Calsequestrin. Additional evidences of Calsequestrin depolymerization were found in imaging studies of Calsequestrin structure in mouse myofibers (Manno et al., this meeting). Supported by NIH grants AR049184 and GM111254.
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Two-Edged Sword: The Ca2+ Biosensor D4cpv-Calsequestrin Restores Functionality to Calsequestrin-Null Muscle
Biophysical Journal, 2012Co-Authors: Monika Sztretye, Carlo Manno, Paul D. Allen, Eduardo RíosAbstract:SR Ca2+ buffering power, BP, decreases during Ca2+-depleting depolarizations of mouse skeletal muscle. During Ca2+ release the stage of high BP is characterized by a “hump” in the release flux waveform. After the depolarization BP returns slowly to its initial value, as demonstrated by the absence of a hump in the flux induced by the second pulse of pairs separated by 600 ms. These time-dependent features were described as “buffer hysteresis” and shown to be contributed by Calsequestrin in the Sztretye et al., companion poster. SR release flux and BP were measured in Calsequestrin 1-null cells expressing the biosensor D4cpv-Calsequestrin. Null cells had lower BP and generally lacked the hump in the flux. In some regions of these cells, however, [biosensor] reached very high values. The spatial heterogeneity of expression permitted comparison of flux and buffer properties evaluated with the same depolarizing pulse simultaneously in regions of widely different [biosensor]. The hump of Ca2+ release flux and other features of buffer hysteresis were restored in regions of Calsequestrin-null cells where D4cpv-Calsequestrin reached above 8 μmol/liter of cytosol. The restoration was partial or nil at [biosensor] below 3 μM. At 10 μM a functional Calsequestrin moiety in the biosensor should provide 800 μM binding sites (or 400 μM Ca2+ at 50% occupancy), a significant contribution compared with the estimates of amount released (1240 μM in WT, 867 μM in the null). Therefore, in addition to a targeted biosensor D4cpv-Calsequestrin is a fluorescently tagged Ca2+ buffer. Moreover, the restoration of hysteretic Ca2+ buffering features indicates that this biosensor contributes the full buffer functionality of Calsequestrin. To our knowledge, this is the first example of a molecule with the functional properties of both a biosensor and a native protein.Funded by NIAMS/NIH.
Marek Michalak - One of the best experts on this subject based on the ideXlab platform.
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Phylogenetic and biochemical analysis of Calsequestrin structure and association of its variants with cardiac disorders.
Scientific reports, 2020Co-Authors: Qian Wang, Tautvydas Paskevicius, Alexander Filbert, Qin Wenying, Hyeong Jin Kim, Xing-zhen Chen, Jingfeng Tang, Joel B. Dacks, Luis B. Agellon, Marek MichalakAbstract:Calsequestrin is among the most abundant proteins in muscle sarcoplasmic reticulum and displays a high capacity but a low affinity for Ca2+ binding. In mammals, Calsequestrin is encoded by two genes, CASQ1 and CASQ2, which are expressed almost exclusively in skeletal and cardiac muscles, respectively. Phylogenetic analysis indicates that Calsequestrin is an ancient gene in metazoans, and that the duplication of the ancestral Calsequestrin gene took place after the emergence of the lancelet. CASQ2 gene variants associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) in humans are positively correlated with a high degree of evolutionary conservation across all Calsequestrin homologues. The mutations are distributed in diverse locations of the Calsequestrin protein and impart functional diversity but remarkably manifest in a similar phenotype in humans.
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Calsequestrin. Structure, function, and evolution
Cell calcium, 2020Co-Authors: Qian Wang, Marek MichalakAbstract:Calsequestrin is the major Ca2+ binding protein in the sarcoplasmic reticulum (SR), serves as the main Ca2+ storage and buffering protein and is an important regulator of Ca2+ release channels in both skeletal and cardiac muscle. It is anchored at the junctional SR membrane through interactions with membrane proteins and undergoes reversible polymerization with increasing Ca2+ concentration. Calsequestrin provides high local Ca2+ at the junctional SR and communicates changes in luminal Ca2+ concentration to Ca2+ release channels, thus it is an essential component of excitation-contraction coupling. Recent studies reveal new insights on Calsequestrin trafficking, Ca2+ binding, protein evolution, protein-protein interactions, stress responses and the molecular basis of related human muscle disease, including catecholaminergic polymorphic ventricular tachycardia (CPVT). Here we provide a comprehensive overview of Calsequestrin, with recent advances in structure, diverse functions, phylogenetic analysis, and its role in muscle physiology, stress responses and human pathology.
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Regulation of calcium binding proteins calreticulin and Calsequestrin during differentiation in the myogenic cell line L6.
Journal of cellular physiology, 1996Co-Authors: Suzanne Tharin, Marek Michalak, Paul A. Hamel, Edward M. Conway, Michal OpasAbstract:In this report we defined the structural and temporal limits within which calreticulin and Calsequestrin participate in the muscle cell phenotype, in the L6 model myogenic system. Calreticulin and Calsequestrin are two Ca2+ binding proteins thought to participate in intracellular Ca2+ homeostasis. We show that Calsequestrin protein and mRNA were expressed when L6 cells were induced to differentiate, during which time the level of expression of calreticulin protein did not change appreciably. Calreticulin mRNA levels, however, were constant throughout L6 cell differentiation except for slight decline in the mRNA levels at the very late stages of L6 differentiation (day 11-12). We also show that the two Ca2+ binding proteins are coexpressed in differentiated L6 cells. Based on its mobility in SDS-PAGE, L6 rat skeletal muscle cells in culture expressed cardiac isoform of Calsequestrin. In the mature rat skeletal muscle, calreticulin and Calsequestrin were localized to sarcoplasmic reticulum (SR). Calreticulun, but not Calsequestrin, staining was also observed in the perinuclear region. These data suggest that expression of calreticulin and Calsequestrin may be under different control during myogenesis in rat L6 cells in culture.
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Zn2+ binding to cardiac Calsequestrin.
Biochemical and biophysical research communications, 1995Co-Authors: Shairaz Baksh, Cornelia Spamer, Kimio Oikawa, William D. Mccubbin, Claus Heilmann, Cyril M. Kay, Marek MichalakAbstract:Abstract Zn2+ binding to canine cardiac Calsequestrin was investigated using the Zn2+ specific fluorescence dye salicylcarbohydrazone (SACH), 65Zn2+ overlay and Zn2+-IDA chromatography. Cardiac Calsequestrin binds ∼200 moles of Zn2+/mole of protein with the Kd=300 μM. Zn2+ binding to Calsequestrin was further confirmed by 65Zn2+ overlay and Zn2+-dependent aggregation of the protein. However, Calsequestrin did not bind to a Zn2+-IDA-agarose column, indicating that histidine residues may not be involved in Zn2+ binding to the protein. Circular dichroism revealed only minor Zn2+-dependent conformational changes in Calsequestrin. We conclude that Calsequestrin is a Ca2+- and Zn2+-binding protein and that Zn2+ may modulate the structure and function of the protein.
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calreticulin and not Calsequestrin is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum
Journal of Biological Chemistry, 1991Co-Authors: Rachel E. Milner, Michal Opas, Shairaz Baksh, C Shemanko, M R Carpenter, L Smillie, J E Vance, Marek MichalakAbstract:Abstract The distribution of Calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-Calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-Calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding Calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which Calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of Calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not Calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.
Magdolna Varsányi - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of skeletal muscle Calsequestrin enhances its Ca2+ binding capacity and promotes its association with junctin.
Cell calcium, 2008Co-Authors: Nicole A. Beard, Magdolna Varsányi, Lan Wei, Stephanie N. Cheung, Takashi Kimura, Angela F. DulhuntyAbstract:Summary Calcium signaling, intrinsic to skeletal and cardiac muscle function, is critically dependent on the amount of calcium stored within the sarcoplasmic reticulum. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the ryanodine receptor and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. We examined the influence of phosphorylation of Calsequestrin on its ability to store calcium, to polymerise and to regulate ryanodine receptors by binding to triadin and junctin. Our hypothesis was that these parameters might be altered by phosphorylation of threonine 353, which is located near the calcium and triadin/junctin binding sites. Although phosphorylation increased the calcium binding capacity of Calsequestrin nearly 2-fold, it did not alter Calsequestrin polymerisation, its binding to triadin or junctin or inhibition of ryanodine receptor activity at 1 mM luminal calcium. Phosphorylation was required for Calsequestrin binding to junctin when calcium concentration was low (100 nM), and ryanodine receptors were activated by dephosphorylated Calsequestrin when it bound to triadin alone. These novel data shows that phosphorylated Calsequestrin is required for high capacity calcium buffering and suggest that ryanodine receptor inhibition by Calsequestrin is mediated by junctin.
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The conformation of Calsequestrin determines its ability to regulate skeletal ryanodine receptors.
Biophysical Journal, 2006Co-Authors: Magdolna Varsányi, Angela F. Dulhunty, Nicole A. BeardAbstract:Ca2+ efflux from the sarcoplasmic reticulum decreases when store Ca2+ concentration falls, particularly in skinned fibers and isolated vesicles where luminal Ca2+ can be reduced to very low levels. However ryanodine receptor activity in many single channel studies is higher when the luminal free Ca2+ concentration is reduced. We investigated the hypothesis that prolonged exposure to low luminal Ca2+ causes conformational changes in Calsequestrin and deregulation of ryanodine receptors, allowing channel activity to increase. Lowering of luminal Ca2+ from 1 mM to 100 μM for several minutes resulted in conformational changes with dissociation of 65–75% of Calsequestrin from the junctional face membrane. The Calsequestrin remaining associated no longer regulated channels. In the absence of this regulation, ryanodine receptors were more active when luminal Ca2+ was lowered from 1 mM to 100 μM. In contrast, when ryanodine receptors were Calsequestrin regulated, lowering luminal Ca2+ either did not alter or decreased activity. Ryanodine receptors are regulated by Calsequestrin under physiological conditions where Calsequestrin is polymerized. Since depolymerization occurs slowly, Calsequestrin can regulate the ryanodine receptor and prevent excess Ca2+ release when the store is transiently depleted, for example, during high frequency activity or early stages of muscle fatigue.
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regulation of ryanodine receptors by Calsequestrin effect of high luminal ca2 and phosphorylation
Biophysical Journal, 2005Co-Authors: Nicole A. Beard, Magdolna Varsányi, Derek R Laver, Marco G Casarotto, Angela F. DulhuntyAbstract:Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that Calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which Calsequestrin dissociates from the ryanodine receptor and the effect of Calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of ≥4 mM dissociates Calsequestrin from junctional face membrane, whereas in the range of 1–3 mM Calsequestrin remains attached; 2), the association with Calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of Calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that Calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.
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Surface plasmon resonance studies prove the interaction of skeletal muscle sarcoplasmic reticular Ca2+ release channel/ryanodine receptor with Calsequestrin
FEBS letters, 2000Co-Authors: Anke Herzog, Csaba Szegedi, Istvan Jona, Friedrich W. Herberg, Magdolna VarsányiAbstract:A high affinity molecular interaction is demonstrated between Calsequestrin and the sarcoplasmic reticular Ca2+ release channel/ryanodine receptor (RyR) by surface plasmon resonance. KD values of 92 nM and 102 nM for the phosphorylated and dephosphorylated Calsequestrin have been determined, respectively. Phosphorylation of Calsequestrin seems not to influence this high affinity interaction, i.e. Calsequestrin might always be bound to RyR. However, the phosphorylation state of Calsequestrin determines the amount of Ca2+ released from the lumen. Dephosphorylation of approximately 1% of the phosphorylated Calsequestrin could be enough to activate the RyR channel half-maximally, as we have shown previously [Szegedi et al., Biochem. J. 337 (1999) 19].
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Calsequestrin: More than 'only' a luminal Ca2+ buffer inside the sarcoplasmic reticulum
Biochemical Journal, 1998Co-Authors: Csaba Szegedi, Anke Herzog, Istvan Jona, Sándor Sárközi, Magdolna VarsányiAbstract:In striated muscle, the sarcoplasmic reticulum (SR) Ca2+ release/ryanodine receptor (RyR) channel provides the pathway through which stored Ca2+ is released into the myoplasm during excitation-contraction coupling. Various luminal Ca2+-binding proteins are responsible for maintaining the free [Ca2+] at 10(-3)-10(-4) M in the SR lumen; in skeletal-muscle SR, it is mainly Calsequestrin. Here we show that, depending on its phosphorylation state, Calsequestrin selectively controls the RyR channel activity at 1 mM free luminal [Ca2+]. Calsequestrin exclusively in the dephosphorylated state enhanced the open probability by approx. 5-fold with a Hill coefficient (h) of 3.3, and increased the mean open time by about 2-fold, i.e. solely dephosphorylated Calsequestrin regulates Ca2+ release from the SR. Because Calsequestrin has been found to occur mainly in the phosphorylated state in the skeletal-muscle SR for the regulation of RyR channel activity, the dephosphorylation of Calsequestrin would appear to be a quintessential physiological event.
