Identification and characterization of three calmodulin binding sites of the skeletal muscle ryanodine receptor.
In the present study, we have identified calmodulin binding sequences in the skeletal muscle ryanodine receptor Ca2+ release channel. Ligand overlays on RYR fusion proteins indicate that the skeletal muscle RYR contains three calmodulin binding regions defined by residues 2937-3225, 3546-3655, and 4425-4621. The RYR fusion protein PC28 (residues 2937-3225) bound calmodulin in the presence of EGTA and Ca2+, while RYR fusion protein PC26 (residues 3546-3655) exhibited strong calmodulin binding at 10 microM Ca2+. The RYR fusion protein PC15 (residues 4425-4621) did not bind calmodulin in the presence of either EGTA or 10-50 microM Ca2+. In the presence of 100-500 microM Ca2+, the RYR fusion protein PC15 exhibited an affinity for calmodulin of approximately 50 nM. Peptides RYR1 PM2 (residues 3610-3629) and RYR1 PM3 (4534-4552) encompassing putative RYR-calmodulin binding sites were synthesized. The synthetic peptides interacted directly with dansylcalmodulin as demonstrated by their capacity to affect the fluorescence emission of dansylcalmodulin. Missense mutation analysis indicates that the Lys and Arg residues are essential for calmodulin binding to the synthetic peptide RYR1 PM3. The RYR calmodulin binding site defined by peptide PM3 lies in the myoplasmic loop 2, a few residues upstream of the putative transmembrane segment M5; the other two calmodulin binding sites are next to the putative transmembrane segments M' and M''. Thus, the effect of calmodulin on Ca2+ release might involve the regulation of the putative transmembrane segments M5, M', and M''.
Menegazzi
P
P
Istituto di Patologia Generale, Universita degli Studi di Ferrara, Italy.
Larini
F
F
Treves
S
S
Guerrini
R
R
Quadroni
M
M
Zorzato
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D015220:Calcium Channels; D002147:Calmodulin; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D000252:Calcium-Transporting ATPases