Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregate Myopathy

Lewis, K.M.; Ronish, Leslie A.; Rı́os, Eduardo; Kang, ChulHee

doi:10.1074/jbc.m115.686261

Cited by 28 publications

(68 citation statements)

References 24 publications

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“…Buffering power B, the incremental ratio of bound over free ligand concentration, is maximal at zero ligand concentration, when the buffer is ligand-free, for most buffers. The buffering power of calsequestrin measured in vitro instead increases with the concentrations of free and bound calcium (6). This increase in buffering ability occurs because the loci for most calcium binding are at the interfaces between calsequestrin monomers, requiring two monomers to achieve useful binding affinity (12).…”

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confidence: 99%

“…Depolymerization would derail the hypothetical diffusional enhancement by destroying the calcium wires; it might also allow migration of calsequestrin away from triads, with damaging targeting and functional repercussions. Finally, all calsequestrin mutations associated with diseases of skeletal muscle (6) and many of the known calsequestrin mutations linked to cardiac diseases (17) are known or predicted to alter its polymerization; therefore, it can be anticipated that any changes that hinder normal polymerization of this protein [including the posttranslational modifications recently studied (18)]…”

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confidence: 99%

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Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

Manno

Figueroa

Gillespie

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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 [Ca 2+ ] 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 [Ca 2+ ] 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.skeletal muscle | excitation/contraction coupling | cardiac muscle | muscle diseases | catecholaminergic polymorphic ventricular tachycardia

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confidence: 99%

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confidence: 99%

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

Manno

Figueroa

Gillespie

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…17; a full list is included in supplemental Table 1). These proteins with highly charged binding sites share common biological functions for conformational switching or calcium titration; for example, calsequestrin (58), human Hsp70 (59), and an Na ϩ /Ca 2ϩ exchanger binding domain (60) have a similarly high charge density binding pocket as Protein S. Many of these proteins with exceptionally electronegative binding sites have already been reported to harbor great structural instability in the absence of calcium (54,55,(57)(58)(59)(60)(61)(62) or have increases in folding speed due to calcium (63). Because many of these proteins are also multidomain, our results on Protein S may apply to many of the proteins with high charge density sites.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein

Scholl¹,

Li²,

Yang

et al. 2016

Journal of Biological Chemistry

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Although multidomain proteins predominate the proteome of all organisms and are expected to display complex folding behaviors and significantly greater structural dynamics as compared with single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands are poorly understood due to a lack of experimental techniques. The multidomain calcium-binding ␤␥-crystallin proteins are particularly important because their deterioration and misfolding/aggregation are associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding ␤␥-crystallin protein, Protein S, and elaborate on its interactions with calcium. We ask whether domain interactions and calcium binding affect Protein S folding and potential structural heterogeneity. Our results from single-molecule force spectroscopy show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca 2؉ , which is mechanically stable in contrast to other proteins that were observed to sample a molten globule under similar conditions. Mutagenesis experiments and computer simulations reveal that the alternative conformation of the N-terminal domain is caused by structural instability produced by the high charge density of a calcium binding site. We find that this alternative conformation in the N-terminal domain is diminished in the presence of calcium and can also be partially eliminated with a hitherto unrecognized compensatory mechanism that uses the interaction of the C-terminal domain to neutralize the electronegative site. We find that up to 1% of all identified multidomain calcium-binding proteins contain a similarly highly charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand.Multidomain proteins are highly prevalent in the proteomes of all organisms (1); however, there is still little known about their folding pathways and interaction with ligands (2), as compared with small, single-domain proteins. This is because large, multidomain proteins may exhibit significant structural dynamics and transient interactions between the domains that are rather difficult to capture and analyze. An important example of this class of proteins are the calcium-binding proteins that are effectors for stimulating cellular signals (3, 4) and buffering calcium levels (5). Currently, of particular interest are the calcium-binding ␤␥-crystallin proteins that are implicated in several human diseases (6 -8). The complexity of these proteins may best be understood through single-molecule methods that are able to deconvolve their conformational heterogeneity. Although there have been exciting recent single-molecule studies of the multidomain calmodulin, including the impact of domains' interactions on calcium binding (9), and studies on mechanical unfolding of ␤␥-crystallins that captured their interesting domain swapping behavior (10), ther...

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“…From a functional point of view, muscle fibers isolated from these patients show (i) decreased Ca 2+ release following caffeine administration, (ii) increased glycogen content and misoriented SR junctions, (iii) an increase by approximately 25% of calsequestrin 1 content and (iv) a lower content of polymerized calsequestin [35,73]. Interestingly a report on the physicochemical properties of wild type and p.D244G mutated CASQ1 showed that the mutation reduces the Ca 2+ binding properties of calsequestrin 1 and causes it to form large aggregates in vitro [36]. It should be pointed out that all the patients identified so far were heterozygous for the causative CASQ1 mutation, while the functional studies were performed on homozygous mutated calsequestrin 1 molecules.…”

Section: Casq1 Mutations and Vacuolar Aggregate Myopathymentioning

confidence: 98%

“…Two isoforms of calsequestrin have been identified: calsequestrin1 that is predominantly expressed in fast twitch skeletal muscles and calsequestrin 2 that is expressed in cardiac and slow twitch muscle. Mutations in CSQ2 (the gene encoding calsequestrin 2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), sudden cardiac arrest and heart failure [33,34] and will not be discussed in this review, whereas mutations in CSQ1 (the gene encoding calsequestrin 1) have been implicated in rare cases of malignant hyperthermia and vacuolar aggregate myopathy [35,36].…”

Section: Excitation-contraction Couplingmentioning

confidence: 99%

Ca2+ handling abnormalities in early-onset muscle diseases: Novel concepts and perspectives

Treves

Jungbluth

Voermans

et al. 2017

Seminars in Cell & Developmental Biology

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a b s t r a c tThe physiological process by which Ca 2+ is released from the sarcoplasmic reticulum is called excitationcontraction coupling; it is initiated by an action potential which travels deep into the muscle fiber where it is sensed by the dihydropyridine receptor, a voltage sensing L-type Ca 2+ channel localized on the transverse tubules. Voltage-induced conformational changes in the dihydropyridine receptor activate the ryanodine receptor Ca 2+ release channel of the sarcoplasmic reticulum. The released Ca 2+ binds to troponin C, enabling contractile thick-thin filament interactions. The Ca 2+ is subsequently transported back into the sarcoplasmic reticulum by specialized Ca 2+ pumps (SERCA), preparing the muscle for a new cycle of contraction. Although other proteins are involved in excitation-contraction coupling, the mechanism described above emphasizes the unique role played by the two Ca 2+ channels (the dihydropyridine receptor and the ryanodine receptor), the SERCA Ca 2+ pumps and the exquisite spatial organization of the membrane compartments endowed with the proteins responsible for this mechanism to function rapidly and efficiently. Research over the past two decades has uncovered the fine details of excitation-contraction coupling under normal conditions while advances in genomics have helped to identify mutations in novel genes in patients with neuromuscular disorders. While it is now clear that many patients with congenital muscle diseases carry mutations in genes encoding proteins directly involved in Ca 2+ homeostasis, it has become apparent that mutations are also present in genes encoding for proteins not thought to be directly involved in Ca 2+ regulation. Ongoing research in the field now focuses on understanding the functional effect of individual mutations, as well as understanding the role of proteins not specifically located in the sarcoplasmic reticulum which nevertheless are involved in Ca 2+ regulation or excitation-contraction coupling. The principal challenge for the future is the identification of drug targets that can be pharmacologically manipulated by small molecules, with the ultimate aim to improve muscle function and quality of life of patients with congenital muscle disorders. The aim of this review is to give an overview of the most recent findings concerning Ca 2+ dysregulation and its impact on muscle function in patients with congenital muscle disorders due to mutations in proteins involved in excitation-contraction coupling and more broadly on Ca 2+ homeostasis.

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Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregate Myopathy

Cited by 28 publications

References 24 publications

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein

Ca2+ handling abnormalities in early-onset muscle diseases: Novel concepts and perspectives

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