title: Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin.
abstract: Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Fruen
authors.forename: Edward M
authors.initials: EM
authors.lastname: Balog
authors.forename: Janet
authors.initials: J
authors.lastname: Schafer
authors.forename: Florentin R
authors.initials: FR
authors.lastname: Nitu
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D018482:Muscle, Skeletal / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000502:physiology; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
chemical_list: D002147:Calmodulin; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C038518:acrylodan; D015081:2-Naphthylamine; D002118:Calcium