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

Date

2001

Authors

Dulhunty, Angela
Laver, Derek Rowland
Curtis, Suzanne
Pace, Suzy M
Haarmann, Claudia
Gallant, Esther

Journal Title

Journal ISSN

Volume Title

Publisher

Biophysical Society

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 Ca2+. 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 CaMKIIβ 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 Ca2+.

Description

Keywords

Keywords: acid phosphatase; adenosine; adenosine triphosphate; adenosine triphosphate derivative; calcium ion; cyclic AMP dependent protein kinase; n [2 [[n [3 (4 chlorophenyl) 2 propenyl] n methylamino]methyl]phenyl] n (2 hydroxyethyl) 4 methoxybenzenesulfonamide;

Citation

Source

Biophysical Journal

Type

Journal article

Book Title

Entity type

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DOI

Restricted until

2037-12-31