Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca 2+ storage protein essential for SR Ca 2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca 2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca 2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca 2+ leak, resulting in premature spontaneous SR Ca 2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca 2+ storage, lack of Casq2 also causes increased diastolic SR Ca 2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

A comprehensive discussion is presented of advances in understanding the structure and function of phospholamban (PLB), the principal regulator of the Ca2+-ATPase of cardiac sarcoplasmic reticulum. Extensive historical studies are reviewed to provide perspective on recent developments. Phospholamban gene structure, expression, and regulation are presented in addition to in vitro and in vivo studies of PLB protein structure and activity. Applications of breakthrough experimental technologies in identifying PLB structure-function relationships and in defining its interaction with the Ca2+-ATPase are also highlighted. The current leading viewpoint of PLB's mechanism of action emerges from a critical examination of alternative hypotheses and the most recent experimental evidence. The potential physiological relevance of PLB function in human heart failure is also covered. The interest in PLB across diverse biochemical disciplines portends its continued intense scrutiny and its potential exploitation as a therapeutic target.

Several key proteins have been localized to junctional sarcoplasmic reticulum which are important for Ca 2؉ release. These include the ryanodine receptor, triadin, and calsequestrin, which may associate into a stable complex at the junctional membrane. We recently purified and cloned a fourth component of this complex, junctin, which exhibits homology with triadin and is the major 125 I-calsequestrin-binding protein detected in cardiac sarcoplasmic reticulum vesicles (Jones, L. R., Zhang, L., Sanborn, K., Jorgensen, A. O., and Kelley, J. (1995) J. Biol. Chem. 270, 30787-30796). In the present study, we have examined the binding interactions between the cardiac forms of these four proteins with emphasis placed on the role of junctin. By a combination of approaches including calsequestrin-affinity chromatography, filter overlay, immunoprecipitation assays, and fusion protein binding analyses, we find that junctin binds directly to calsequestrin, triadin, and the ryanodine receptor. This binding interaction is localized to the lumenal domain of junctin, which is highly enriched in charged amino acids organized into "KEKE" motifs. KEKE repeats are also found in the common lumenal domain of triadin, which likewise is capable of binding to calsequestrin and the ryanodine receptor (Guo, W., and Campbell, K. P. (1995) J. Biol. Chem. 270, 9027-9030). It appears that junctin and triadin interact directly in the junctional sarcoplasmic reticulum membrane and stabilize a complex that anchors calsequestrin to the ryanodine receptor. Taken together, these results suggest that junctin, calsequestrin, triadin, and the ryanodine receptor form a quaternary complex that may be required for normal operation of Ca 2؉ release.

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.