Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca2+ release in skeletal muscle

Abstract

The mechanisms that terminate Ca2+ release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca2+ concentration inside the sarcoplasmic reticulum (SR), [Ca2+]SR, simultaneously with that in the cytosol, [Ca2+]c, during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca2+ release flux (derived from [Ca2+]c(t)) over the gradient that drives it (essentially equal to [Ca2+]SR) provided directly, for the first time, a dynamic measure of the permeability to Ca2+ of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca2+]SR stabilized at ∼35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ∼10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca2+ release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca2+]SR, sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca2+ release.

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

“…Sztretye et al (20) showed that long-lasting membrane depolarization in myofibers under voltage clamp causes only partial calcium store depletion; their finding is in agreement with the present evidence that depolymerization may be graded and remains limited during normal muscle function. The same work showed that depletion is partial because RyR channels close before the SR is fully depleted.…”

“…In all other experiments illustrated in Fig. 1A, the fast calcium buffer BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] was present in the internal solution to reduce calcium-dependent inactivation of the RyR channels (20). The presence of BAPTA increases the flux of calcium release and results in a sharper hump, which can be revealed using much briefer pulses, in turn, making elaborate kinetic studies feasible.…”

Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregate Myopathy

“…This termination, which is essential for rapid contractile relaxation, requires fast closing of SR Ca 2ϩ release (RyR) channels. Some evidence indi-cates that Casq is required for adequate channel closure in skeletal muscle (13,14), but the issue remains controversial (15). Much of the interest in the properties of this protein stems from observations of linkage between its mutations and human disease (special cases of "couplonopathies" or diseases of the couplon (19)).…”

Compartment calcium model of frog skeletal muscle during activation

“…This is similar to cardiac myocytes where SR and cytosolic calcium concentrations play a role in calcium induced calcium release (CICR) from the SR via ryanodine receptors (RyRs, Sobie et al, 2002;Sobie and Lederer, 2012). Similar to cardiac myocytes, recent studies have also shown that calsequestrin, a Ca 2 þ binding protein in the SR, may also help regulate termination or inactivation of Ca 2 þ release from the SR (Berchtold et al, 2000;Ikemoto et al, 1989;Kawasaki and Kasai, 1994;Sztretye et al, 2011).…”