Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca 2؉ ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca 2؉ binding curves were measured using atomic absorption spectroscopy. This method enables highly accurate measurements even for Ca 2؉ bound to polymerized protein. The binding curves for both skeletal and cardiac calsequestrin were complex, with binding increases that correlated with protein dimerization, tetramerization, and oligomerization. The Ca 2؉ binding capacities of skeletal and cardiac calsequestrin are directly compared for the first time, with ϳ80 Ca 2؉ ions bound per skeletal calsequestrin and ϳ60 Ca 2؉ ions per cardiac calsequestrin, as compared with net charges for these molecules of ؊80 and ؊69, respectively. Deleting the negatively charged and disordered C-terminal 27 amino acids of cardiac calsequestrin results in a 50% reduction of its calcium binding capacity and a loss of Ca 2؉ -dependent tetramer formation. Based on the crystal structures of rabbit skeletal muscle calsequestrin and canine cardiac calsequestrin, Ca 2؉ binding capacity data, and previous lightscattering data, a mechanism of Ca 2؉ binding coupled with polymerization is proposed.Calsequestrin (CSQ) 1 binds and releases large quantities of Ca 2ϩ through its high capacity (40 -50 mol of Ca 2ϩ ion per molecule) and relatively low affinity interactions with Ca 2ϩ (K d ϭ 1 mM) (1). Because of this Ca 2ϩ -buffering capacity of CSQ in the lumenal space, the concentration of free Ca 2ϩ in the sarcoplasmic reticulum (SR) can be maintained below the inhibitory level of the Ca 2ϩ pump (1 mM), and simultaneously, the SR can maintain the ability to rapidly deliver a high capacity Ca 2ϩ signal to the cytoplasm. Even though the lumenal space is minuscule compared with the extracellular space, the high concentrations (ϳ100 mg/ml) of CSQ make the SR an efficient storage compartment for Ca 2ϩ (2).CSQ is associated physically with the RyR protein by a nucleation event that involves CSQ binding to the basic lumenal domains of triadin (3) or junctin (4). These two proteins interact with RyR in the junctional face region of the SR, and this network of interacting proteins assures that high concentrations of Ca 2ϩ are stored very near to the site of Ca 2ϩ release. Ca 2ϩ release from CSQ through the Ca 2ϩ release channel is regulatory but not limiting.The Ca 2ϩ binding and dissociation mechanisms of CSQ are not yet clearly understood. Ca 2ϩ binding sites in CSQ are supposed to be very different from those in the Ca 2ϩ pump (sarco(endo)plasmic reticulum calcium ATPase (SERCA)), calmodulin, and troponin C. CSQ sites need to be made and broken but not over the low cytosolic Ca 2ϩ concentration range or with the same stoichiometry and precision as those formed and subsequently disrupted in the Ca 2ϩ pump or...