Calsequestrin Is an Inhibitor of Skeletal Muscle Ryanodine Receptor Calcium Release Channels

Abstract

We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.

Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

“…CSQ not only binds Ca 2+ but also regulates Ca 2+ release by communicating with the RyR via two intermediary proteins, triadin and junctin (Beard et al, 2002;Gyorke et al, 2004;Wei et al, 2006), which are found in many tissues and play a ubiquitous role in Ca 2+ signaling. Both are transmembrane proteins that bind to CSQ and the RyR (Jones et al, 1995) to form a CSQ/ triadin/junctin/RyR "luminal Ca 2+ transduction machine" that is central to Ca 2+ release unit function (Beard et al, 2002(Beard et al, , 2004Gyorke et al, 2004;Wei et al, 2006) and assembly (Tijskens et al, 2003).…”

“…However, since the bulk of the protein is located within the SR lumen where it binds to CSQ and the RyR (Knudson et al, 1993b;Guo and Campbell, 1995), it is currently believed to facilitate cross-talk between CSQ and the RyR (Beard et al, 2002), rather than directly infl uence EC coupling (Gyorke et al, 2004). Junctin was later discovered and thought to have a comparable function to triadin, due to its similar structure and ability to also bind CSQ and the RyR (Jones et al, 1995;Zhang et al, 1997;Tijskens et al, 2003).…”

Fast kinetics of calcium dissociation from calsequestrin

“…Yet, when using 1 mM free [Ca 2+ ] in the trans (luminal) compartment, the single-channel activity of native RyRmeasured in conditions that presumably allow RyR incorporation into lipid bilayers with CSQ, triadin, and junctin -is maximally inhibited by CSQ. In contrast, under the same conditions, the purified skeletal RyR is not inhibited (Beard et al, 2002). These results have been interpreted as evidence of RyR inhibition by CSQ, which would be attached to the RyR channels in bilayers in the presence of 1 mM luminalfree [Ca 2+ ] (Beard et al, 2002).…”

“…In contrast, under the same conditions, the purified skeletal RyR is not inhibited (Beard et al, 2002). These results have been interpreted as evidence of RyR inhibition by CSQ, which would be attached to the RyR channels in bilayers in the presence of 1 mM luminalfree [Ca 2+ ] (Beard et al, 2002). Since in 1 mM Ca 2+ CSQ is expected to have a significant α-helical content, CSQ seems to affect differently native RyR when forming part of isolated SR vesicles than when incorporated in bilayers.…”

Redox-assisted regulation of Ca 2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5

“…4A, Right). (18,19). Calsequestrin consists of three tandem repeats of thioredoxin-like domains, which are similar to a part of ERdj5, and was reported to convert its molecular conformation from a monomer to an oligomer depending on [Ca 2+ ], and this conversion affects the interaction with the ryanodine receptor (20,21).…”