Characteristics of irreversible ATP activation suggest that native skeletal ryanodine receptors can be phosphorylated via an endogenous CaMKII.
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+).
Dulhunty
A F
AF
Muscle Research Group, John Curtin School of Medical Research, Canberra, ACT 2601, Australia. angela.dulhunty@anu.edu.au
Laver
D
D
Curtis
S M
SM
Pace
S
S
Haarmann
C
C
Gallant
E M
EM
D000135:Acid Phosphatase / Q000737:chemistry; D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D000483:Alleles; D000818:Animals; D001667:Binding, Competitive; D002110:Caffeine / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D015151:Immunoblotting; D008024:Ligands; D008051:Lipid Bilayers / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013552:Swine; D013997:Time Factors
D000697:Central Nervous System Stimulants; D008024:Ligands; D008051:Lipid Bilayers; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D000266:Adenylyl Imidodiphosphate; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002110:Caffeine; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000135:Acid Phosphatase; D000241:Adenosine