title: Ligand-dependent conformational changes in the clamp region of the cardiac ryanodine receptor.
abstract: Global conformational changes in the three-dimensional structure of the Ca(2+) release channel/ryanodine receptor (RyR) occur upon ligand activation. A number of ligands are able to activate the RyR channel, but whether these structurally diverse ligands induce the same or different conformational changes in the channel is largely unknown. Here we constructed a fluorescence resonance energy transfer (FRET)-based probe by inserting a CFP after residue Ser-2367 and a YFP after residue Tyr-2801 in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (RyR2(Ser-2367-CFP/Tyr-2801-YFP)). Both of these insertion sites have previously been mapped to the "clamp" region in the four corners of the square-shaped cytoplasmic assembly of the three-dimensional structure of RyR2. Using this novel FRET probe, we monitored the extent of conformational changes in the clamp region of RyR2(Ser-2367-CFP/Tyr-2801-YFP) induced by various ligands. We also monitored the extent of Ca(2+) release induced by the same ligands in HEK293 cells expressing RyR2(Ser-2367-CFP/Tyr-2801-YFP). We detected conformational changes in the clamp region for the ligands caffeine, aminophylline, theophylline, ATP, and ryanodine but not for Ca(2+) or 4-chloro-m-cresol, although they all induced Ca(2+) release. Interestingly, caffeine is able to induce further conformational changes in the clamp region of the ryanodine-modified channel, suggesting that ryanodine does not lock RyR in a fixed conformation. Our data demonstrate that conformational changes in the clamp region of RyR are ligand-dependent and suggest the existence of multiple ligand dependent RyR activation mechanisms associated with distinct conformational changes.
authors.affiliation: Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Xixi
authors.initials: X
authors.lastname: Tian
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.forename: Terence
authors.initials: T
authors.lastname: Wagenknecht
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D002118:Calcium