Triadin/Junctin Double Null Mouse Reveals a Differential Role for Triadin and Junctin in Anchoring CASQ to the jSR and Regulating Ca2+ Homeostasis

Abstract

Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca2+ imaging and Ca2+ selective microelectrodes we found that changes in e-c coupling, SR Ca2+content and resting [Ca2+] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca2+ regulation than Jct/CASQ association.

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

“…3F and Table 1). A value of 1 for f ′ð0Þ corresponds to D = 0.05 μm The low estimates of D and the observation that tagged Casq1 is partly immobile agree with the consensus that Casq1 constitutes the dense polymeric network visible inside TC in EM images (9,27). The detailed similarity in structure of calsequestrin polymers in crystals (12) and in vivo (34) provides additional evidence and supports the extrapolation to cells of calsequestrin properties observed in solution, including the close association between polymerization and calcium binding.…”

“…Because the mutants show altered calcium release timing, calsequestrin is believed to modulate gating of ryanodine receptor (RyR) release channels. Because it forms long polymeric tendrils [also known as calcium wires (8)] ending near the SR channel mouth (9), calsequestrin is proposed to lead calcium toward the channel (8,10), a speculation that assumes (as first done in ref. 11) that reduction of dimensionality enhances diffusion.…”

“…Thin sections were imaged after staining with lead acetate. The techniques are detailed by Boncompagni et al (9). Morphometry (determination of areas and lengths of membrane intersections) was done with ImageJ (NIH).…”

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

“…We have interpreted this observation, together with crystallographic studies, as evidence that Casq binds Ca 2ϩ cooperatively, which explains both its greater oligomerization and greater Ca 2ϩ binding capacity as Ca 2ϩ concentrations increase. In vivo, inside the SR, Casq exists in linear ramified polymers, forming a dense network that fills the SR terminal cisternae (6,7). The structure of this network is entirely consistent with the lattice interactions observed in vitro (8).…”

The Ejtm Specials “Mobility in Elderly”

“…Her many contributions to the study of the muscle membrane systems and ability to attract young brilliant scientists to electron microscopy are well known and demonstrated also by two speakers of the 2014Spring PaduaMuscleDays, Feliciano Protasi and Simona Boncompagni of Chieti University. 14,15 They have been and are strongly contributing to the success of FES for permanently denervated muscles. 16 We would like to add to Clara's many merits, the pioneering electron microscopy study in the field of muscle denervation: her 1963 article "An electron microscope study of denervation atrophy in red and white skeletal muscle fibers".…”