Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1.
Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.
Balog
Edward M
EM
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA. balog004@tc.umn.edu
Norton
Laura E
LE
Bloomquist
Rachel A
RA
Cornea
Razvan L
RL
Black
D J
DJ
Louis
Charles F
CF
Thomas
David D
DD
Fruen
Bradley R
BR
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine; D013058:Mass Spectrometry; D008715:Methionine; D010084:Oxidation-Reduction; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine
D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D005973:Glutamine; D008715:Methionine; D002118:Calcium