Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle
Authors: Manno Carlo , Sztretye Monika , Figueroa Lourdes , Allen Paul D. , Ríos Eduardo 
Published: November 2012

Key points
The rise in cytosolic calcium ion concentration that triggers muscle contraction requires release of a large amount of calcium from the cellular store, sarcoplasmic reticulum (SR), where it is stored bound, largely to the protein calsequestrin.
Binding of calcium by calsequestrin is a complex process, believed to involve changes in protein conformation and aggregation. We want to know to what extent these properties, observed in vitro, apply inside cells.
We measured the calcium buffering power of the SR, defined as the ratio of change in total SR calcium by change in free [Ca2+]SR, in muscle cells of wild type or calsequestrin‐lacking mice, using two different methods to monitor [Ca2+]SR and deriving changes in total SR calcium content from simultaneous measurements of cytosolic [Ca2+].
The average buffering power during a large, depleting calcium release event was 157 in the wild type and 40 in the calsequestrin‐null mice, suggesting that three‐quarters of the calcium released normally comes from the calsequestrin‐bound pool.
The Ca2+ buffering ability of the SR is different from that of the equivalent concentration of calsequestrin in aqueous solution, the SR exhibiting greater affinity and cooperativity. We conclude that calsequestrin adopts different properties inside cells.
SR buffering power depends on the SR Ca2+ load and on the rate of its changes, a dependence that could be, at least in part, explained by the unique Ca2+ binding properties of calsequestrin.
This study reveals Ca2+ buffering as a highly dynamic process, marking it as both a vulnerable link in diseases that involve loss of control of Ca2+ release, and a candidate for further study and intervention.
Abstract  The buffering power, B, of the sarcoplasmic reticulum (SR), ratio of the changes in total and free [Ca2+], was determined in fast‐twitch mouse muscle cells subjected to depleting membrane depolarization. Changes in total SR [Ca2+] were measured integrating Ca2+ release flux, determined with a cytosolic [Ca2+] monitor. Free [Ca2+]SR was measured using the cameleon D4cpv‐Casq1. In 34 wild‐type (WT) cells average B during the depolarization (ON phase) was 157 (SEM 26), implying that of 157 ions released, 156 were bound inside the SR. B was significantly greater when BAPTA, which increases release flux, was present in the cytosol. B was greater early in the pulse – when flux was greatest – than at its end, and greater in the ON than in the OFF. In 29 Casq1‐null cells, B was 40 (3.6). The difference suggests that 75% of the releasable calcium is normally bound to calsequestrin. In the nulls the difference in B between ON and OFF was less than in the WT but still significant. This difference and the associated decay in B during the ON were not artifacts of a slow SR monitor, as they were also found in the WT when [Ca2+]SR was tracked with the fast dye fluo‐5N. The calcium buffering power, binding capacity and non‐linear binding properties of the SR measured here could be accounted for by calsequestrin at the concentration present in mammalian muscle, provided that its properties were substantially different from those found in solution. Its affinity should be higher, or KD lower than the conventionally accepted 1 mm; its cooperativity (n in a Hill fit) should be higher and the stoichiometry of binding should be at the higher end of the values derived in solution. The reduction in B during release might reflect changes in calsequestrin conformation upon calcium loss.