The Role of Calsequestrin, Triadin, and Junctin in Conferring Cardiac Ryanodine Receptor Responsiveness to Luminal Calcium

Abstract

The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…However, there have been conflicting results regarding luminal Ca 2þ regulation and the source of these discrepancies is unclear. These might reflect altered mechanisms because of different experimental conditions, for example, differences in membrane preparation that results in variation of the protein complexes formed between RyR and calsequestrin, triadin, and junctin that are known to regulate RyR via luminal Ca 2þ (Györke et al 2004). Mg 2þ is believed to inhibit RyRs by two mechanisms: reducing RyR open probability by competing with high-affinity Ca 2þ activation sites, or by binding to less selective low-affinity Ca 2þ sites that also mediate Ca 2þ inhibition Laver et al 1997).…”

Abnormal intrastore calcium signaling in chronic heart failure

“…[Ca] inside the SR is an important determinant of the functional activity of the RyR Ca release channels (5,26). Normally, RyR luminal Ca regulation appears to play a stabilizing role by countering the intrinsic positive feedback of Ca-induced Ca release during the release process.…”

“…RyR open probability changes as a direct function of [Ca] at the luminal side of the channel (3,4). The responsiveness of RyRs to luminal Ca seems to be mediated by the auxiliary proteins triadin, junctin, and calsequestrin (CASQ2), which are coupled to RyRs at the luminal surface of the SR (5). During the release process, the increase in cytosolic Ca is accompanied by a simultaneous, reciprocal decline in intra-SR Ca (6)(7)(8).…”

Calcium Mobilization via Intracellular Ion Channels, Store Organization and Mitochondria in Smooth Muscle

“…The close coupling of dihydropyridine receptors and RyR provides control of Ca 2+ release by Ca 2+ influx. The quaternary complexes between triadin, junctin, RyR, and CSQ provides the luminal Ca 2+ sensing capabilities that regulates RyR activity [85].…”