Fast kinetics of calcium dissociation from calsequestrin

Abstract

We measured the kinetics of calcium dissociation from calsequestrin in solution or forming part of isolated junctional sarcoplasmic reticulum membranes by mixing calsequestrin equilibrated with calcium with calcium-free solutions in a stopped-flow system. In parallel, we measured the kinetics of the intrinsic fluorescence changes that take place following calcium dissociation from calsequestrin. We found that at 25ºC calcium dissociation was 10-fold faster for calsequestrin attached to junctional membranes (k = 109 s -1 ) than in solution. These results imply that calcium dissociation from calsequestrin in vivo is not rate limiting during excitation-contraction coupling. In addition, we found that the intrinsic fluorescence decrease for calsequestrin in solution or forming part of junctional membranes was significantly slower than the rates of calcium dissociation. The kinetics of intrinsic fluorescence changes had two components for calsequestrin associated to junctional membranes and only one for calsequestrin in solution; the faster component was 8-fold faster (k = 54.1 s -1 ) than the slower component (k = 6.9 s -1 ), which had the same k value as for calsequestrin in solution. These combined results suggest that the presence of calsequestrin at high concentrations in a restricted space, such as when bound to the junctional membrane, accelerates calcium dissociation and the resulting structural changes, presumably as a result of cooperative molecular interactions.

Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast‐ and slow‐twitch fibres of rat

“…The present study offers a new perspective on one of the major roles of CSQ1 in fast-twitch muscle. By necessity, fast-twitch fibres need to have a substantial amount of Ca 2+ stored in the SR that can be released rapidly upon stimulation (Beltrán et al 2006) in order to saturate the Ca 2+ -binding sites on troponin C. They must also have a high density of SERCA pumps in order to be able to rapidly resequester the Ca 2+ for fast relaxation. The high density of SERCA pumps not only increases the number of cytoplasmic Ca 2+ -binding sites and consequent Ca 2+ release requirement, but also greatly increases the number of leak pathways out of the SR.…”

Ca2+-Mobility in the Sarcoplasmic Reticulum of Ventricular Myocytes Is Low

“…4 D) was solved by best-fitting a single D CaSR value in the diffusion model. Depletion can be fitted in this way because [Ca 21 ] SR equilibrates relatively rapidly (;10 ms) with [Ca 21 ] SRT (30), and the two parameters will be linearly related, given that the binding constant (3,30,31) of the principal luminal buffer, calsequestrin, is greater than or similar to [Ca 21 ] SR . Fitting results are shown in Fig.…”

Dantrolene sodium increases calcium binding by human recombinant cardiac calsequestrin and calcium loading by sheep cardiac sarcoplasmic reticulum

“…Since DaNa appears to facilitate CASQ2 polymerization and enhanced Ca 2+ binding to CASQ2 at lower [Ca 2+ ] it was important to verify that DaNa increases the SR Ca 2+ storage and makes more Ca 2+ available for subsequent SR Ca 2+ release. When the free [Ca 2+ ] decreases, CASQ2 undergoes depolymerization and Ca 2+ dissociates at a faster rate than the Ca 2+ release from the SR, with CASQ2 thus providing localized intraSR Ca 2+ storage for easy release …”