title: Localization of an NH(2)-terminal disease-causing mutation hot spot to the "clamp" region in the three-dimensional structure of the cardiac ryanodine receptor.
abstract: A region between residues 414 and 466 in the cardiac ryanodine receptor (RyR2) harbors more than half of the known NH(2)-terminal mutations associated with cardiac arrhythmias and sudden death. To gain insight into the structural basis of this NH(2)-terminal mutation hot spot, we have determined its location in the three-dimensional structure of RyR2. Green fluorescent protein (GFP), used as a structural marker, was inserted into the middle of this mutation hot spot after Ser-437 in the RyR2 sequence. The resultant GFP-RyR2 fusion protein, RyR2(S437-GFP,) was expressed in HEK293 cells and characterized using Ca(2+) release, [(3)H]ryanodine binding, and single cell Ca(2+) imaging studies. These functional analyses revealed that RyR2(S437-GFP) forms a caffeine- and ryanodine-sensitive Ca(2+) release channel that possesses Ca(2+) and caffeine dependence of activation indistinguishable from that of wild type (wt) RyR2. HEK293 cells expressing RyR2(S437-GFP) displayed a propensity for store overload-induced Ca(2+) release similar to that in cells expressing RyR2-wt. The three-dimensional structure of the purified RyR2(S437-GFP) was reconstructed using cryo-electron microscopy and single particle image processing. Subtraction of the three-dimensional reconstructions of RyR2-wt and RyR2(S437-GFP) revealed the location of the inserted GFP, and hence the NH(2)-terminal mutation hot spot, in a region between domains 5 and 9 in the clamp-shaped structure. This location is close to a previously mapped central disease-causing mutation site located in a region between domains 5 and 6. These results, together with findings from previous studies, suggest that the proposed interactions between the NH(2)-terminal and central regions of RyR2 are likely to take place between domains 5 and 6 and that the clamp-shaped structure, which shows substantial conformational differences between the closed and open states, is highly susceptible to disease-causing mutations.
authors: Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors: Ruiwu
authors: R
authors: Wang
authors: Wenqian
authors: W
authors: Chen
authors: Shitian
authors: S
authors: Cai
authors: Jing
authors: J
authors: Zhang
authors: Jeff
authors: J
authors: Bolstad
authors: Terence
authors: T
authors: Wagenknecht
authors: Zheng
authors: Z
authors: Liu
authors: S R Wayne
authors: SR
authors: Chen
mesh_terms: D000818:Animals; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism; D002110:Caffeine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D020285:Cryoelectron Microscopy; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D012694:Serine; D002118:Calcium