Phosphorylation with protein kinases modulates calcium loading of terminal cisternae of sarcoplasmic reticulum from skeletal muscle

• Published in: Cell calcium (Edinburgh) 1995-09, Vol.18 (3), p.197-206
• Main Authors: Mayrleitner, M., Chandler, R., Schindler, H., Fleischer, S.
• Format: Article
• Language: English
• Subjects: Adenylyl Imidodiphosphate - pharmacology; Animals; Calcium - metabolism; Calcium Channels - metabolism; Calcium-Transporting ATPases - metabolism; Ion Transport - physiology; Magnesium - physiology; Muscle Fibers, Fast-Twitch - enzymology; Muscle Fibers, Fast-Twitch - metabolism; Muscle Proteins - metabolism; Phosphoprotein Phosphatases - pharmacology; Phosphorylation; Protein Kinases - metabolism; Protein Phosphatase 1; Rabbits; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum - metabolism; Space life sciences
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1016/0143-4160(95)90064-0

We previously found in single channel studies that ryanodine receptor (RyR) channel activity can be made insensitive to block by Mg 2+ when terminal cisternae of sarcoplasmic reticulum, incorporated into planar bilayers, are treated with protein kinase A (PKA) or Ca 2+/calmodulin dependent protein kinase type II (CamPK II), and then again made sensitive by treatment with protein phosphatases [Hain J. Nath S. Mayrleitner M. Fleischer S. Schindler H. (1994) Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. Biophys. J., 67, 1823–1833]. In this study, modulation by protein kinases and phosphatases on net Ca 2+ uptake by TC is presented. Phosphorylation of TC vesicles with PKA, CamPK II, or protein kinase C (PKC) reduced the calcium loading rate of TC vesicles 3-fold, 2.1-fold and 1.7-fold, respectively, measured in the presence of 1 MM MgCl 2. There is no effect when AMP-PNP is substituted for ATP. Phosphorylation of the RyR was also measured by incororation of [γ- 32P]-phosphate from ATP. A phosphorylation stoichiometry of 1.94 ± 0.1 ( 32P/RyR) for PKA, 0.89 ± 0.08 for CamPK II and 0.95 ± 0.16 for PKC was obtained under these conditions. A study of the time dependence of phosphorylation with PKA and CamPK shows a direct correlation of reduction in calcium loading rate with increased phosphorylation of the ryanodine receptor. Treatment with protein phosphatase 1 enhanced the calcium loading rate again, after it was reduced by PKA phosphorylation. Investigation of the magnesium dependency shows that even at higher [Mg 2+] (6 mM), PKA phosphorylated TC vesicles have a 2.3-fold reduced calcium loading rate indicating insensitivity to block by Mg 2+. Protein kinases have no effect on Ca 2+-ATPase activity in TC or on Ca 2+ loading in longitudinal tubules which are devoid of RyR. We interpret the decreased net Ca 2+ uptake as being due to enhanced Ca 2+ leakage via the RyR. These studies show, at a macroscopic level, that the calcium release channel is modulated by kinases/phosphatases and support our previous findings with single channel studies that the ryanodine receptor must be phosphorylated to be active under physiological Mg 2+ concentrations.