Characteristics of Irreversible ATP Activation Suggest that Native Skeletal Ryanodine Receptors Can Be Phosphorylated via an Endogenous CaMKII

Abstract

Phosphorylation of skeletal muscle ryanodine receptor (RyR) calcium release channels by endogenous kinases incorporated into lipid bilayers with native sarcoplasmic reticulum vesicles was investigated during exposure to 2 mM cytoplasmic ATP. Activation of RyRs after 1-min exposure to ATP was reversible upon ATP washout. In contrast, activation after 5 to 8 min was largely irreversible: the small fall in activity with washout was significantly less than that after brief ATP exposure. The irreversible activation was reduced by acid phosphatase and was not seen after exposure to nonhydrolyzable ATP analogs. The data suggested that the channel complex was phosphorylated after addition of ATP and that phosphorylation reduced the RyR's sensitivity to ATP, adenosine, and Ca(2+). The endogenous kinase was likely to be a calcium calmodulin kinase II (CaMKII) because the CaMKII inhibitor KN-93 and an inhibitory peptide for CaMKII prevented the phosphorylation-induced irreversible activation. In contrast, phosphorylation effects remained unchanged with inhibitory peptides for protein kinase C and A. The presence of CaMKIIbeta in the SR vesicles was confirmed by immunoblotting. The results suggest that CaMKII is anchored to skeletal muscle RyRs and that phosphorylation by this kinase alters the enhancement of channel activity by ATP and Ca(2+).

Roles of the calcineurin and CaMK signaling pathways in fast-to-slow fiber type transformation of cultured adult mouse skeletal muscle fibers

“…Calcineurin has been shown to respond to the slow fiber-type pattern of electrical stimulation and thus to require elevated [Ca 2ϩ ] i (6,13). In contrast, other studies show that [Ca 2ϩ ] i as low as 100 nM can activate CaMKII and induce phosphorylation of RyR complexes (9,14,17). Furthermore, autophosphorylation of CaMKII in the presence of Ca 2ϩ -CaM can cause it to remain active independent of Ca 2ϩ -CaM for a long period (5,22).…”

The recombinant dihydropyridine receptor II–III loop and partly structured ‘C’ region peptides modify cardiac ryanodine receptor activity

“…In addition, unrelated peptides, luteinizinghormone-releasing hormone (A. F. Dulhunty and S. M. Curtis, unpublished work) and inhibitory peptides for protein kinase A, protein kinase C and Ca 2+ /calmodulin kinase II [32], all fail to alter RyR activity.…”

“…It is not clear whether the DHPR fragments bind to the RyR2 or to proteins that remain associated with the cytoplasmic side of the channel, such as FK506-binding proteins, Ca 2+ /calmodulin kinase II (on RyR1) or protein kinase A (on RyR2) [32,[41][42][43]. However, the binding sites for the DHPR fragments are most likely to be on RyR2, since binding to the protein is seen in two-hybrid [4] and surface plasmon resonance studies [3].…”

Characterization of Recombinant Skeletal Muscle (Ser-2843) and Cardiac Muscle (Ser-2809) Ryanodine Receptor Phosphorylation Mutants

“…Metabolic labeling of RyR2 provided direct evidence for additional phosphorylation sites by showing that CaMKII phosphorylates at least four additional sites in RyR (16). In support of a physiological role, CaMKII co-purified with cardiac muscle RyR (17) and, in skeletal SR vesicles, remained anchored to RyR in lipid bilayer measurements, altering the regulation of channel activity by ATP and Ca 2ϩ (18). In skeletal muscle, ␣KAP, a non-kinase protein, has been identified as one of the anchoring proteins necessary for targeting the CaMKII holoenzyme to the SR membrane (19).…”