Comparing Skeletal and Cardiac Calsequestrin Structures and Their Calcium Binding

Abstract

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...

Depletion “skraps” and dynamic buffering inside the cellular calcium store

“…Ca 2ϩ favors formation of these polymers and stabilizes them, occupying a layer where it is adsorbed rather than bound (20). In this layer, Ca 2ϩ is able to diffuse laterally and should be readily deliverable to the open channels (20). According to the properties of CSQ in aqueous solutions, this polymer-specific bound Ca 2ϩ could constitute the proximate store, evolving as illustrated in Fig.…”

“…Bonds with triadin and junctin (22) place CSQ polymers near the luminal mouth of RyRs. Ca 2ϩ favors formation of these polymers and stabilizes them, occupying a layer where it is adsorbed rather than bound (20). In this layer, Ca 2ϩ is able to diffuse laterally and should be readily deliverable to the open channels (20).…”

“…6E). In the well loaded SR, CSQ is polymerized (20). During Ca 2ϩ release, [Ca 2ϩ ] SR will decrease near release sites, which should cause widespread depolymerization of CSQ (20) with loss of binding sites (20) and release of Ca 2ϩ to the SR lumen (Fig.…”

“…It follows that the initial surge in [Ca 2ϩ ] SR resulted from a decrease in buffering power, namely in the number or affinity of Ca 2ϩ binding sites of the store buffer. This change is expected from CSQ, because this protein polymerizes and gains Ca 2ϩ binding sites in the presence of elevated [Ca 2ϩ ], whereas the opposite occurs as [Ca 2ϩ ] is lowered (20).…”

Transcriptional Analysis of the Human Cardiac Calsequestrin Gene in Cardiac and Skeletal Myocytes

“…At this time there are no studies that point to differences in the physiological role of both CASQ isoforms, thus their roles shall be considered equivalent. The rabbit CASQ1 and the dog CASQ2 have been crystallized (6,7); results of crystallization studies shown that the casq monomer was found to be constituted by three almost identical domains (I, II, and III) similar to that of Escherichia coli thioredoxin domain. It was found that CASQ polymerizes in response to rising concentrations of Ca 2ϩ in the lumen of the SR, to form a homotetrameric complex (10 M to 1 mM) and at higher concentrations of polymers, in concentrations higher than 10 mM the CASQ polymer dissociates from the Ca 2ϩ releasing channel (8).…”