title: FRET detection of calmodulin binding to the cardiac RyR2 calcium release channel.
abstract: Calmodulin (CaM) binding to the type 2 ryanodine receptor (RyR2) regulates Ca release from the cardiac sarcoplasmic reticulum (SR). However, the structural basis of CaM regulation of the RyR2 is poorly defined, and the presence of other potential CaM binding partners in cardiac myocytes complicates resolution of CaM's regulatory interactions with RyR2. Here, we show that a fluorescence-resonance-energy-transfer (FRET)-based approach can effectively resolve RyR2 CaM binding, both in isolated SR membrane vesicles and in permeabilized ventricular myocytes. A small FRET donor was targeted to the RyR2 cytoplasmic assembly via fluorescent labeling of the FKBP12.6 subunit. Acceptor fluorophore was attached at discrete positions within either the N- or the C-lobe of CaM. FRET between FKBP12.6 and CaM bound to SR vesicles indicated CaM binding at a single high-affinity site within 60 Å of FKBP12.6. Micromolar Ca increased the apparent affinity of CaM binding and slowed CaM dissociation, but did not significantly affect maximal FRET efficiency at saturating CaM. FRET was strongest when the acceptor was attached at either of two positions within CaM's N-lobe versus sites in CaM's C-lobe, providing CaM orientation information. In permeabilized ventricular myocytes, FKBP12.6 and CaM colocalized to Z-lines, and the efficiency of energy transfer to both the N- and C-lobes of CaM was comparable to that observed in SR vesicle experiments. Results also indicate that both the location and orientation of CaM binding on the RyR2 are very similar to the skeletal muscle RyR1 isoform. Specific binding of CaM to functional RyR2 channels in the cardiac myocyte environment can be monitored using FKBP biosensors and FRET.
authors: Department of Pharmacology, University of California, Davis, California, USA.
authors: Tao
authors: T
authors: Guo
authors: Bradley R
authors: BR
authors: Fruen
authors: Florentin R
authors: FR
authors: Nitu
authors: Trinh D
authors: TD
authors: Nguyen
authors: Yi
authors: Y
authors: Yang
authors: Razvan L
authors: RL
authors: Cornea
authors: Donald M
authors: DM
authors: Bers
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D008164:Luminescent Proteins / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D013498:Suramin / Q000494:pharmacology; D034421:Sus scrofa; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D002147:Calmodulin; D008164:Luminescent Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D013498:Suramin; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus