scholarly journals Multiple regions of junctin drive interaction with calsequestrin-1 and localization at triads in skeletal muscle

2021 ◽  
Author(s):  
Daniela Rossi ◽  
Stefania Lorenzini ◽  
Enrico Pierantozzi ◽  
Filip Van Petegem ◽  
David Osamwonuyi Amadsun ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Ryanodine Receptor ◽  
Transmembrane Protein ◽  
Sr Proteins ◽  
Receptor Interaction ◽  
Striated Muscles ◽  
Charged Amino Acids ◽  
Multiple Regions ◽  
Junctional Sarcoplasmic Reticulum

Junctin is a transmembrane protein of striated muscles, localized at the junctional sarcoplasmic reticulum (j-SR). It is characterized by a luminal C-terminal tail, through which it functionally interacts with calsequestrin and the ryanodine receptor. Interaction with calsequestrin was ascribed to the presence of stretches of charged amino acids. However, the regions able to bind calsequestrin have not been defined in detail. We report here that, in non-muscle cells, junctin and calsequestrin assemble in long linear regions within the endoplasmic reticulum, mirroring the formation of calsequestrin polymers. In differentiating myotubes, the two proteins co-localize at triads, where they assemble with other j-SR proteins. By performing GST pull-down assays with distinct regions of the junctin tail, we identified two KEKE motifs able to bind calsequestrin. In addition, stretches of charged amino acids downstream these motifs were found to be also able to bind calsequestrin and the ryanodine receptor. Deletion of even one of these regions impaired the ability of junctin to localize at the j-SR, suggesting that interaction with other proteins at this site represents a key element in junctin targeting.

Identification of the A-band localization domain of myosin binding proteins C and H (MyBP-C, MyBP-H) in skeletal muscle

1999 ◽  
Vol 112 (1) ◽  
pp. 69-79 ◽  
Author(s):  
R. Gilbert ◽  
J.A. Cohen ◽  
S. Pardo ◽  
A. Basu ◽  
D.A. Fischman
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Binding Proteins ◽  
Thick Filament ◽  
Terminal Segment ◽  
Binding Domain ◽  
Striated Muscles ◽  
Fibronectin Type Iii ◽  
Chimeric Construct ◽  
Myosin Binding

Although major constituents of the thick filaments of vertebrate striated muscles, the myosin binding proteins (MyBP-C and MyBP-H) are still of uncertain function. Distributed in the cross-bridge bearing zone of the A-bands of myofibrils, in a series of transverse 43 nm stripes, the proteins are constructed of a tandem series of small globular domains, each composed of approximately 90–100 amino acids, which have sequence similarities to either the C2-set of immunoglobulins (IgC2) and the fibronectin type III (FnIII) motifs. MyBP-C is composed of ten globular domains (approximately 130 kDa) whereas MyBP-H is smaller (approximately 58 kDa) and consists of a unique N-terminal segment followed by four globular domains, the order of which is identical to that of MyBP-C (FnIII-IgC2-FnIII-IgC2). To improve our understanding of this protein family we have characterized the domains in each of these two proteins which are required for targeting the proteins to their native site(s) in the sarcomere during myogenesis. Cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding mutant constructs of the MyBPs bearing an N-terminal myc epitope, and their localization to the A-band examined by immunofluorescence microscopy. Based on the clarity and intensity of the myc A-band signals we concluded that constructs encoding the four C-terminal motifs of MyBP-C and MyBP-H (approximately 360 amino acids) were all that was necessary to efficiently localize each of these peptides to the A-band. Truncation mutants lacking one of these 4 domains were less efficiently targeted to the C-zone of the sarcomere. Deletion of the last C-terminal motif of MyBP-H, its myosin binding domain, abolished all localization to the A-band. A chimeric construct, HU-3C10, in which the C-terminal motif of MyBP-H was replaced by the myosin binding domain of MyBP-C, efficiently localized to the A-band. Taken together, these observations indicate that MyBP-C and MyBP-H are localized to the A-band by the same C-terminal domain, composed of two IgC2 and two FnIII motifs. A model has been proposed for the interaction and positioning of the MyBPs in the thick filament through a ternary complex of the four C-terminal motifs with the myosin rods and titin.

scholarly journals Negatively Charged Amino Acids within the Intraluminal Loop of Ryanodine Receptor Are Involved in the Interaction with Triadin

2003 ◽  
Vol 279 (8) ◽  
pp. 6994-7000 ◽  
Author(s):  
Jae Man Lee ◽  
Seong-Hwan Rho ◽  
Dong Wook Shin ◽  
Chunghee Cho ◽  
Woo Jin Park ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ryanodine Receptor ◽  
Charged Amino Acids

scholarly journals A simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor

1992 ◽  
Vol 285 (1) ◽  
pp. 61-64 ◽  
Author(s):  
V Shoshan-Barmatz ◽  
A Zarka
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Binding Sites ◽  
Specific Activity ◽  
Fast Method ◽  
Step Method ◽  
High Affinity ◽  
Sds Page ◽  
One Step ◽  
Junctional Sarcoplasmic Reticulum

In this paper we describe a simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor. The ryanodine receptor from CHAPS-solubilized junctional sarcoplasmic-reticulum membranes was adsorbed to a spermine-agarose column and eluted by 2 mM-spermine. The purified receptor, consisting predominantly of a 450 kDa polypeptide on SDS/PAGE, binds [3H]ryanodine with a specific activity of approximately 300 pmol/mg of protein and with a high affinity (KD = 4.7 +/- 2 nM). The purified receptor appears to retain the pharmacological properties of the receptor in the original membranes. The purification resulted in over 80% recovery of the initial ryanodine-binding sites and about 30-96-fold purification. This simple and fast method is highly reproducible and suitable for purification of small as well as large quantities of ryanodine receptor.

Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin

2004 ◽  
Vol 287 (5) ◽  
pp. H2216-H2225 ◽  
Author(s):  
Uwe Kirchhefer ◽  
Hideo A. Baba ◽  
Gabriela Hanske ◽  
Larry R. Jones ◽  
Paulus Kirchhof ◽  
...  
Keyword(s):  
Heart Rate ◽  
Transgenic Mice ◽  
Ryanodine Receptor ◽  
Transmembrane Protein ◽  
Force Of Contraction ◽  
Stress Tests ◽  
Age Dependent ◽  
Beating Rate ◽  
Right Atria ◽  
Junctional Sarcoplasmic Reticulum

Junctin is a transmembrane protein of the cardiac junctional sarcoplasmic reticulum (SR) that binds to the ryanodine receptor, calsequestrin, and triadin 1. This quaternary protein complex is thought to facilitate SR Ca2+ release. To improve our understanding of the contribution of junctin to the regulation of SR function, we examined the age-dependent effects of junctin overexpression in the atrium of 3-, 6-, and 18-wk-old transgenic mice. The ratio of atrial weight and body weight was unchanged between junctin-overexpressing (JCN) and wild-type (WT) mice at all ages investigated ( n = 6–8). The protein expression of triadin 1 was decreased starting in 3-wk-old JCN atria (by 69%), whereas the expression of the ryanodine receptor was diminished in 6- (by 48%) and 18-wk-old (by 57%) JCN atria compared with age-matched WT atria. Force of contraction was decreased by 35% in 18-wk-old JCN compared with age-matched WT left atrial muscle strips, which was accompanied by a prolonged time of relaxation (48.1 ± 0.9 vs. 44.2 ± 0.8 ms, respectively, n = 6–8, P < 0.05). The spontaneous beating rate of isolated right atria was higher in 18-wk-old JCN mice compared with age-matched WT mice (389 ± 10 vs. 357 ± 6 beats/min, respectively, n = 6–8, P < 0.05). Heart rate was lower by 9% in telemetric ECG recordings in 18-wk-old JCN mice during stress tests. Three-week-old JCN atria exhibited a higher potentiation of force of contraction at rest pauses of 30 s (by 13%) and of 300 s (by 35%), suggesting increased SR Ca2+ content. This was consistent with the higher force of contraction in 3-wk-old JCN atria (by 29%) compared with age-matched WT atria (by 10%) under the administration of caffeine. We conclude that in 3-wk-old atria, junctin overexpression was associated with a reduced expression of triadin 1 resulting in a higher SR Ca2+ load without changes in contractility or heart rate. In 6-wk-old JCN atria, the compensatory downregulation of the ryanodine receptor may offset the effects of junctin overexpression. Finally, the progressive decrease in ryanodine receptor density may contribute to the decreased atrial contractility and lower heart rate during stress in 18-wk-old JCN mice.

scholarly journals A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e43817 ◽  
Author(s):  
Elize Wium ◽  
Angela F. Dulhunty ◽  
Nicole A. Beard
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Receptor Interaction ◽  
Interaction Domain

Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1)

2001 ◽  
Vol 360 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Guo Guang DU ◽  
Hideto OYAMADA ◽  
Vijay K. KHANNA ◽  
David H. MacLENNAN
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Atp Binding ◽  
Regulatory Domain ◽  
Wild Type ◽  
Release Channel ◽  
Binding Motifs ◽  
Channel Opening

Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370–2375 and 3937–3940) in the Ca2+-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [3H]ryanodine binding by Ca2+, caffeine and ATP (adenosine 5′-[β,γ-methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca2+ release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [3H]ryanodine-binding properties and EC50 values for Ca2+ activation of high-affinity [3H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1mM AMP-PCP, Ca2+-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370–2375 and 3937–3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370–2375 lie within a sequence (amino acids 2163–2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163–2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1.

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