Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor.
Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) have been linked to certain types of cardiac arrhythmias and sudden death. Two mutation hotspots that lie in the N-terminal and central regions of RyR2 are predicted to interact with one another and to form an important channel regulator switch. To monitor the conformational dynamics involving these regions, we generated a fluorescence resonance energy transfer (FRET) pair. A yellow fluorescent protein (YFP) was inserted into RyR2 after residue Ser437 in the N-terminal region, and a cyan fluorescent protein (CFP) was inserted after residue Ser2367 in the central region, to form a dual YFP- and CFP-labeled RyR2 (RyR2(S437-YFP/S2367-CFP)). We transfected HEK293 cells with RyR2(S437-YFP/S2367-CFP) cDNAs, and then examined them by using confocal microscopy and by measuring the FRET signal in live cells. The FRET signals are influenced by modulators of RyR2, by domain peptides that mimic the effects of disease causing RyR2 mutations, and by various drugs. Importantly, FRET signals were also readily detected in cells co-transfected with single CFP (RyR2(S437-YFP)) and single YFP (RyR2(S2367-CFP)) labeled RyR2, indicating that the interaction between the N-terminal and central mutation regions is an inter-subunit interaction. Our studies demonstrate that FRET analyses of this CFP- and YFP-labeled RyR2 can be used not only for investigating the conformational dynamics associated with RyR2 channel gating, but potentially, also for identifying drugs that are capable of stabilizing the conformations of RyR2.
Liu
Zheng
Z
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA. liuz@wadsworth.org
Wang
Ruiwu
R
Tian
Xixi
X
Zhong
Xiaowei
X
Gangopadhyay
Jaya
J
Cole
Richard
R
Ikemoto
Noriaki
N
Chen
S R Wayne
SR
Wagenknecht
Terence
T
D001145:Arrhythmias, Cardiac / Q000150:complications / Q000235:genetics / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D016757:Death, Sudden, Cardiac / Q000209:etiology; D031541:Fluorescence Resonance Energy Transfer; D020022:Genetic Predisposition to Disease; D006801:Humans; D018613:Microscopy, Confocal; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009206:Myocardium / Q000378:metabolism*; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
D015220:Calcium Channels; D019837:Ryanodine Receptor Calcium Release Channel