CALMODULIN-BINDING SITES OF THE SKELETAL, CARDIAC, AND BRAIN RYANODINE RECEPTOR CA2- MODULATION BY THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE( CHANNELS )

In this study, we define calmodulin binding sites of skeletal, cardiac , and brain ryanodine receptor (RYR) Ca2+ channels. Cardiac and brain RYR peptides corresponding to the calmodulin binding sites present in the skeletal RYR [Menegazzi, P., et al. (1994) Biochemistry 33, 9078-9 084] were synthesized, and their interaction with calmodulin was monit ored by fluorescent techniques. The central portions of the skeletal, cardiac, and brain RYR protomers display one high (CaM1; K-d ranging b etween 2.7 and 10.2 nM) and one low affinity (CaM2; K-d ranging betwee n 116 and 142 nM) calmodulin binding site. Depending on the RYR model having 4 or 12 transmembrane segments, a third calmodulin binding site (CaM3) was identified a few residues upstream from the putative trans membrane segment M1 or M5. Its affinity for calmodulin varied between the RYR isoforms: the cardiac RYR CaM3 displays a high affinity (9.09 +/- 1.0 nM, n = 5), while the skeletal and brain RYR CaM3 have low aff inity, the lowest affinity being displayed by the brain isoform (234 /- 39 nM, n = 3). The RYRs calmodulin binding site CaM1 encompasses th e sequence Arg-His-Arg-Val(Ile)-Ser-Leu, which is phosphorylated in vi tro by the catalytic subunit of the cAMP-dependent protein kinase. Pho sphorylation of RYR PM1 peptides occurs on the Ser, corresponding to a mino acid number 2919, 3020, and 3055 of the brain, cardiac, and skele tal RYR protomers, respectively. We found that phosphorylation of the RYR PM1 peptides was inhibited by calmodulin binding and that the form ation of the PM1 peptide-calmodulin complex was inhibited by peptide p hosphorylation. These data indicate that the effect of calmodulin bind ing to RYR CaM1 may be regulated by the phosphorylation state of the S er residue localized within the sequence Arg-His-Arg-Val(Ile)-Ser-Leu.