Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants.
Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.
Stange
Mirko
M
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, 27599-7260, USA.
Xu
Le
L
Balshaw
David
D
Yamaguchi
Naohiro
N
Meissner
Gerhard
G
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004594:Electrophysiology; D006333:Heart Failure / Q000209:etiology; D006801:Humans; D018482:Muscle, Skeletal / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D012694:Serine / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012694:Serine