Zinc Modulates Skeletal Ryanodine Receptor Function Resulting in Altered Sarcoplasmic Reticulum Calcium Release

Abstract

Changes in cardiac ryanodine receptor (RyR2) phosphorylation are thought to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP1) activity has been observed in heart failure (HF), but the regulatory role of this enzyme on intracellular Ca 2þ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP1 activity, we investigated the effect of the PP1 catalytic subunit on Ca 2þ sparks in permeabilized cardiomyocytes. We used wild-type (WT) and transgenic mice in which the highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation. In WT myocytes, where cytosolic Ca 2þ was clamped at 60nM, 2U/ml of PP1 initially increased Ca 2þ spark frequency (CaSpF) by 2.2-fold, followed by a second phase during which CaSpF returned to control. Spark mass was decreased, but due to the high CaSpF, spark-mediated leak was increased by PP1. This was accompanied by depletion of the sarcoplasmic reticulum (SR) Ca 2þ stores, as determined by application of caffeine. Changes in Ca 2þ release and SR Ca 2þ load were prevented by 5uM of okadaic acid, an inhibitor of PP1. S2808A mutant myocytes showed lower resting CaSpF compared to WT (1.1850.23 vs 2.350.35 sparks/100um/s) and 2U/ml of PP1 failed to generate changes in CaSpF as well as in SR Ca 2þ load. A higher concentration of PP1 (10U/ml) increased CaSpF 4-fold compared to control in WT, and 2.8-fold in S2808A cells, indicating a concentration-dependence. Our results suggest that increased intracellular PP1 activity stimulates RyR2-mediated SR Ca 2þ release and that de-phosphorylation of RyR2-S2808 and at least one not yet identified phosphorylation site may be important in RyR2 modulation.

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics

“…Notably, physiological cytosolic concentrations of Zn 2+ ranging from 100 pM to 1 μM can increase the open probability of the RyR1 channel, while higher concentrations can inhibit ryanodine binding. 24 This dual modulation extends to cardiac muscles, where RyR2 also exhibits zinc concentration dependence, capable of both activating and inhibiting calcium release. Activation likely occurs by binding to the Ca 2+ -activation site, while the inhibitory effect of Zn 2+ may be mediated by the Ca 2+ /Mg 2+ -inhibitory site of RyR1.…”

“…Mutagenesis studies of two ligand residues within the zinc-finger motif have demonstrated their pivotal role in preserving a stable conformation of the RyR1 channel . On the other hand, it has been suggested that Zn 2+ exhibits biphasic modulation in skeletal muscles, similar to calcium, , implying the existence of both activation and inactivation sites. Notably, physiological cytosolic concentrations of Zn 2+ ranging from 100 pM to 1 μM can increase the open probability of the RyR1 channel, while higher concentrations can inhibit ryanodine binding .…”

“…On the other hand, it has been suggested that Zn 2+ exhibits biphasic modulation in skeletal muscles, similar to calcium, , implying the existence of both activation and inactivation sites. Notably, physiological cytosolic concentrations of Zn 2+ ranging from 100 pM to 1 μM can increase the open probability of the RyR1 channel, while higher concentrations can inhibit ryanodine binding . This dual modulation extends to cardiac muscles, where RyR2 also exhibits zinc concentration dependence, capable of both activating and inhibiting calcium release.…”