Functional Interaction of the Cytoplasmic Domain of Triadin with the Skeletal Ryanodine Receptor

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To identify the function of triadin in skeletal muscle, adenovirusmediated overexpression of Trisk 95 or Trisk 51, the two major skeletal muscle isoforms, was induced in rat skeletal muscle primary cultures, and the physiological behavior of the modified cells was analyzed. Overexpression did not modify the expression level of their protein partners ryanodine receptor, dihydropyridine receptor, and the other triadin. Caffeine-induced calcium release was also unaffected by triadin overexpression. Nevertheless, in the absence of extracellular calcium, depolarization-induced calcium release was almost abolished in Trisk 95 overexpressing myotubes (T95 myotubes), and not modified in Trisk 51 overexpressing myotubes (T51 myotubes). This was not because of a modification of dihydropyridine receptors, as depolarization in presence of external calcium still induced a calcium release, and the activation curve of dihydropyridine receptor was unchanged, in both T95 and T51 myotubes. The calcium release complex was also maintained in T95 myotubes as Trisk 95, ryanodine receptor, dihydropyridine receptor, and Trisk 51 were still co-localized. The effect of Trisk 95 overexpression on depolarization-induced calcium release was reversed by a simultaneous infection with an antisense Trisk 95 adenovirus, indicating the specificity of this effect. Thus, the level of Trisk 95 and not Trisk 51 is important on regulating the calcium release complex, and an excess of this protein can lead to an inhibition of the physiological function of the complex. In skeletal muscle cells, excitation-contraction (E-C)2 coupling is the process by which depolarization of the plasma membrane produces a large transient release of Ca 2ϩ from the sarcoplasmic reticulum, which in turn triggers contraction. Dihydropyridine receptors (DHPRs), the L-type Ca 2ϩ channels localized in the T-tubule membrane, serve as the voltage sensors for E-C coupling (1, 2) and activate ryanodine receptors (RyRs), the intracellular Ca 2ϩ release channels of the sarcoplasmic reticulum membrane (3, 4). In skeletal muscle, entry of Ca 2ϩ through DHPR is not required for E-C coupling (5). Instead, a voltage-dependent conformational change in the II-III loop of the skeletal muscle DHPR ␣1 subunit (Cav1.1) activates the skeletal muscle ryanodine receptor, RyR1. Activation of RyR1 results in a release of Ca 2ϩ from the sarcoplasmic reticulum intracellular stores into the cytosol, which in turn activates the cellular contractile apparatus to initiate muscle contraction. In cardiac muscle, the entry of external calcium through DHPR is required to induce activation of RyR and release of internal Ca 2ϩ via RyR, a mechanism known as calcium-induced calcium release. Calcium-induced calcium release is not the initial mechanism responsible of Ca 2ϩ release in skeletal muscle, but it can contribute to the amplification of the signal, and both calcium-induced calcium release and conformational coupling are involved in skeletal muscle contraction, to different extents (6).Triadin is a transmem...

Section: Discussionmentioning

confidence: 50%

“…In skeletal muscle, triadin has been shown to inhibit the calcium channel activity of purified RyR (18,19). These experiments were the first to identify a function of triadin in skeletal muscle, and they led to the conclusion that triadin could regulate RyR function via inhibition of the channel.…”

Section: In Skeletal Muscle Cells Excitation-contraction (E-c)mentioning

confidence: 94%

Triadin (Trisk 95) Overexpression Blocks Excitation-Contraction Coupling in Rat Skeletal Myotubes

Rezgui

Vassilopoulos

Brocard

et al. 2005

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“…Although originally thought to be the link between RyR1 and DHPR (6 -8) and a key modulator of EC coupling (9 -11), it appears evident now that triadin acts primarily as a negative regulator of RyR1 activity (12)(13)(14)(15). Although the mechanism by which triadin regulate RyRs remains unclear, there is support for the hypothesis that triadin is involved in facilitating the cross-communication be-tween calsequestrin (CSQ) and the RyRs (16 -18).…”

mentioning

confidence: 88%

Ablation of Skeletal Muscle Triadin Impairs FKBP12/RyR1 Channel Interactions Essential for Maintaining Resting Cytoplasmic Ca2+

Eltit

Feng

López

et al. 2010

] rest in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca 2؉ entry at rest.In skeletal muscle, where control of cytosolic Ca 2ϩ concentration is key to muscle contraction, the overall Ca 2ϩ homeostasis is preserved by a concerted action of several ionic channels and transporters within the plasma membrane and the sarcoplasmic reticulum (SR).3 These proteins, including plasma membrane Ca 2ϩ ATPase, Na ϩ /Ca 2ϩ exchanger, dihydropyridine receptor, ryanodine receptor (RyRs), and the sarcoendoplasmic reticulum Ca 2ϩ ATPase, among others, interact functionally and physically with each other and with a plethora of regulatory proteins that ultimately modulate their activity, hence, the resting intracellular Ca 2ϩ levels. In muscle cells where SR Ca 2ϩ stores represent the main intracellular sources of Ca 2ϩ release, RyR1 seems positioned to play a key role in regulating myoplasmic [Ca 2ϩ ] rest . An effective example of this can be found in dyspedic cells that lack expression of all isoforms of RyRs (1), where we have recently demonstrated that RyR1 expression was sufficient to remodel the Ca 2ϩ regulatory machinery in a way that ultimately resulted in a significant shift in steady-state Ca 2ϩ fluxes contributing to [Ca 2ϩ ] rest (2). Alterations of RyR1 activity, like those conferred by malignant hyperthermia mutations, also resulted in long term changes in [Ca 2ϩ ] rest (3, 4), raising the possibility that indirect modulation of RyR1 by its protein binding partners may be important for homeostatic regulation of [Ca 2ϩ ] rest . Therefore, finding and understanding the role of RyR1 regulatory proteins could be instrumental in understanding the mechanisms that control long term Ca 2ϩ homeostasis in muscle.Among the endogenous regulators, the RyR1/triadin interactions are particularly intriguing, as its role in skeletal muscle function remains elusive (5). Although originally thought to be the link between RyR1 and DHPR (6 -8) and a key modulator of EC coupling (9 -11), it appears evident now that triadin acts primarily as a negative regulator of RyR1 activity (12-15). Although the mechanism by which triadin regulate RyRs remains unclear, there is support for the hypothesis that triadin is involved in facilitating the cross-communication be-* This work was supported, in whole or in part, by National Institute of Health Grants 5K01AR054818 (to C. F. P.), P01AR47605 (to P. D. A. and I. N. P.), and R01HL076433 (to B. R. F.

“…We do not yet understand the role of HRC, as the more abundant Ca 2ϩ -binding protein calsequestrin appears to serve its role as a Ca 2ϩ store perfectly well. It was shown that calsequestrin plays a role in the regulation of the ryanodine receptor (28,29) in addition to the Ca 2ϩ store function. Thus, HRC may perhaps also have a regulatory role.…”

Section: Discussionmentioning

confidence: 99%

Interaction of HRC (Histidine-rich Ca2+-Binding Protein) and Triadin in the Lumen of Sarcoplasmic Reticulum

Lee

Kang

Kim

et al. 2001

HRC (histidine-rich Ca2؉ binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca 2؉ with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca 2؉ -sensitive. Our data suggest that HRC may play a role in the regulation of Ca 2؉ release from the sarcoplasmic reticulum by interaction with triadin.