MODULATION OF THE SKELETAL-MUSCLE RYANODINE RECEPTOR BY ENDOGENOUS PHOSPHORYLATION OF 160 150-KDA PROTEINS OF THE SARCOPLASMIC-RETICULUM/

This paper demonstrates and characterizes the inhibition of ryanodine binding caused by the phosphorylation of the 160/150-kDa proteins in s keletal muscle sarcoplasmic reticulum (SR). Inhibition of ryanodine bi nding was obtained by preincubation of SR membranes with ATP + NaF. Th e inhibition was characterized by the following findings: (a) If ATP w as replaced by AdoPP[NH]P, inhibition of ryanodine binding activity wa s not observed. (b) The inhibitory effect of preincubation with ATP NaF, like the phosphorylation of 150/160-kDa proteins, was Ca2+ depend ent. (c) Inhibition of ryanodine binding, as the protein phosphorylati on, was not observed if NaF (> 30 mM) was replaced with okadaic acid. (d) The optimal pH for the inhibition and the phosphorylation was abou t 7.0. (e) Both the phosphorylation of the 160/150-kDa proteins and in hibition of ryanodine binding were prevented by dichlorobenzimidazole riboside and hemin, inhibitors of casein kinase II. (f) Dephosphorylat ion of the 160/150-kDa proteins prevented the inhibition of ryanodine binding. (g) The presence of NP-40 during the phosphorylation prevente d both the 160/150-kDa phosphorylation and the inhibition of ryanodine binding. Furthermore, a linear relationship was obtained between the degree of ryanodine binding inhibition and the level of phosphorylatio n of the 160/150-kDa proteins, as controlled by ATP or NaF concentrati ons. The binding affinity for Ca2+ of the ryanodine receptor (RyR) was modified by phosphorylation of the 160/150-kDa proteins, decreasing b y up to 100-fold. The phosphorylation of the SR membranes resulted in an elimination of ryanodine binding sites with slight effect on the ry anodine binding affinity. These results suggest the modulation of the properties of the RyR by phosphorylation/dephosphorylation of the 160/ 150-kDa proteins. The identification of the phosphorylated 160/150-kDa proteins, their kinase, and the structural interactions between them and the RyR are presented in the accompanying paper.