Key result
Insertion of GFP into the RyR2 NH2-terminal mutation hot spot localized this region between domains 5 and 9 in the clamp-shaped structure, close to a central disease-causing mutation site.
The NH2-terminal mutation hot spot in the cardiac ryanodine receptor localizes to the clamp region, providing structural insights into its role in cardiac arrhythmias.
Structural insights into RyR2 mutation hotspot leave open targeted therapies; hypothesis-generating for arrhythmia mechanisms pending clinical validation.
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.
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Wang et al. (2007) studied Cardiac arrhythmias and sudden death (associated with RyR2 mutations). GFP insertion after Ser-437 in RyR2 sequence (RyR2(S437-GFP)) vs. Wild type (wt) RyR2 was evaluated on Location of the NH2-terminal mutation hot spot in the three-dimensional structure of RyR2. Insertion of GFP into the RyR2 NH2-terminal mutation hot spot localized this region between domains 5 and 9 in the clamp-shaped structure, close to a central disease-causing mutation site.