Journal of Biological Chemistry
Volume 295, Issue 46, 13 November 2020, Pages 15622-15635
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Membrane Biology
The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability

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Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.

Ca2+ activation
Mg2+ inhibition
basal activity
ryanodine binding
calcium intracellular release
calcium imaging
ryanodine receptor
sarcoplasmic reticulum (SR)
inositol trisphosphate receptor (InsP3R)
calcium
calcium channel

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Author contributions—W. G., B. S., R. W., and S. W. C. conceptualization; W. G., J. P. E., and R. W. data curation; W. G. and B. S. formal analysis; W. G., B. S., and S. W. C. investigation; W. G., B. S., and R. W. methodology; W. G., R. W., and S. W. C. writing-original draft; B. S., J. P. E., and S. W. C. writing-review and editing; J. P. E. and S. W. C. project administration; S. W. C. resources; S. W. C. supervision; S. W. C. funding acquisition.

Funding and additional information—This work was supported by the Canadian Institutes of Health Research Grant PJT-155940 (to S. R. W. C.), the Heart and Stroke Foundation of Canada Grant G-19-0026444 (to S. R. W. C.), and the Heart and Stroke Foundation Chair in Cardiovascular Research (to S. R. W. C.). W. G. is a recipient of the Alberta Innovates-Health Solutions Graduate Studentship Award, and B. S. is a recipient of the Heart and Stroke Foundation of Canada Junior Fellowship Award and the AIHS Fellowship Award.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

    RyR2

    cardiac ryanodine receptor

    CICR

    Ca2+-induced Ca2+ release

    CTD

    C-terminal domain

    CPVT

    catecholaminergic polymorphic ventricular tachycardia

    KRH

    Krebs-Ringer-Hepes

    ANOVA

    analysis of variance.