Role of calmodulin methionine residues in mediating productive association with cardiac ryanodine receptors.
Calmodulin (CaM) binds to the cardiac ryanodine receptor Ca2+ release channel (RyR2) with high affinity and may act as a regulatory channel subunit. Here we determine the role of CaM Met residues in the productive association of CaM with RyR2, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to cardiac muscle sarcoplasmic reticulum (SR) vesicles. Oxidation of all nine CaM Met residues abolished the productive association of CaM with RyR2. Substitution of the COOH-terminal Mets of CaM with Leu decreased the extent of CaM inhibition of cardiac SR (CSR) vesicle [3H]ryanodine binding. In contrast, replacing the NH2-terminal Met of CaM with Leu increased the concentration of CaM required to inhibit CSR [3H]ryanodine binding but did not alter the extent of inhibition. Site-specific substitution of individual CaM Met residues with Gln demonstrated that Met124 was required for both high-affinity CaM binding to RyR2 and for maximal CaM inhibition. These results thus identify a Met residue critical for the productive association of CaM with RyR2 channels.
Balog
Edward M
EM
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, USA. ed.balog@ap.gatech.edu
Norton
Laura E
LE
Thomas
David D
DD
Fruen
Bradley R
BR
D019943:Amino Acid Substitution; D000818:Animals; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism*; D005973:Glutamine; D066298:In Vitro Techniques; D007930:Leucine; D008715:Methionine / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010210:Papillary Muscles / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013552:Swine
D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D005973:Glutamine; D008715:Methionine; D007930:Leucine