Measurement of RyR Permeability Reveals a Role of Calsequestrin in Termination of SR \(Ca^{2+}\) Release in Skeletal Muscle

Abstract

The mechanisms that terminate \(Ca^{2+}\) 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 \(Ca^{2+}\) concentration inside the sarcoplasmic reticulum (SR), \([Ca^{2+}]_{SR}\), simultaneously with that in the cytosol, \([Ca^{2+}]_c\), during the response to long-lasting depolarization of the plasma membrane. The ratio of \(Ca^{2+}\) release flux (derived from \([Ca^{2+}]_c(t)\)) over the gradient that drives it (essentially equal to \([Ca^{2+}]_{SR}\)) provided directly, for the first time, a dynamic measure of the permeability to \(Ca^{2+}\) of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, \([Ca^{2+}]_{SR}\) 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 \(Ca^{2+}\) 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 \([Ca^{2+}]_{SR}\), sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates \(Ca^{2+}\) release.