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.
Highlights
The sarcoplasmic reticulum (SR) of skeletal muscle is a differentiated domain of the endoplasmic reticulum [1] that acts as the intracellular Ca2+store
The junctional SR (jSR) functionally communicates with invaginations of the surface membrane where RyR1 interacts with several protein components forming functional multi-protein complexes defined as the Calcium Release Unit (CRU)
To assess the effect of the absence of Jct, Tdn and Tdn/Jct on the relative expression levels of several CRU components we examined crude membrane preparations of WT, Tdn-null, Jct-null and Tdn/Jct double-null from hind leg muscles using Western blots analysis
Summary
The sarcoplasmic reticulum (SR) of skeletal muscle is a differentiated domain of the endoplasmic reticulum [1] that acts as the intracellular Ca2+store. The SR has two clearly delimited domains with distinct function, structure and composition: the free SR (fSR) rich in sarco-endoplasmic reticulum Ca2+ ATPase (SERCA1) important for Ca2+ re-uptake and the junctional SR (jSR), containing among other proteins the ryanodine receptor Ca2+ release channels (RyRs) and the Ca2+ binding protein calsequestrin (CASQ). The jSR functionally communicates with invaginations of the surface membrane (the transverse tubules, T-tubule) where RyR1 interacts with several protein components forming functional multi-protein complexes defined as the Calcium Release Unit (CRU). In adult skeletal muscle CRUs are in the form of triads with two jSR cisternae, called lateral sacs, facing a central T-tubule. In the junctional face membrane of the jSR, RyR1 interacts with Tdn, Jct and CASQ forming a macromolecular complex thought to regulate RyR1 activity [2,3,4,5,6]. It is expected that monomeric CASQ is not directly visible in the EM due to its small size and because of this structural observations do not allow for studies of the ratio of polymer versus monomer at a given point in time or on possible cycling between the two states during a contraction cycle [17]
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