The functional significance of redox-mediated intersubunit cross-linking in regulation of human type 2 ryanodine receptor

Roman Nikolaienko, Elisa Bovo, Robyn T. Rebbeck, Daniel Kahn, David D. Thomas, Razvan L. Cornea, Aleksey V. Zima

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

The type 2 ryanodine receptor (RyR2) plays a key role in the cardiac intracellular calcium (Ca2+) regulation. We have previously shown that oxidative stress activates RyR2 in rabbit cardiomyocytes by promoting the formation of disulfide bonds between neighboring RyR2 subunits. However, the functional significance of this redox modification for human RyR2 (hRyR2) remains largely unknown. Here, we studied the redox regulation of hRyR2 in HEK293 cells transiently expressing the ryr2 gene. Analysis of hRyR2 cross-linking and of the redox-GFP readout response to diamide oxidation revealed that hRyR2 cysteines involved in the intersubunit cross-linking are highly sensitive to oxidative stress. In parallel experiments, the effect of diamide on endoplasmic reticulum (ER) Ca2+ release was studied in cells co-transfected with hRyR2, ER Ca2+ pump (SERCA2a) and the ER-targeted Ca2+ sensor R-CEPIA1er. Expression of hRyR2 and SERCA2a produced “cardiac-like” Ca2+ waves due to spontaneous hRyR2 activation. Incubation with diamide caused a fast decline of the luminal ER Ca2+ (or ER Ca2+ load) followed by the cessation of Ca2+ waves. The maximal effect of diamide on ER Ca2+ load and Ca2+ waves positively correlates with the maximum level of hRyR2 cross-linking, indicating a functional significance of this redox modification. Furthermore, the level of hRyR2 cross-linking positively correlates with the degree of calmodulin (CaM) dissociation from the hRyR2 complex. In skeletal muscle RyR (RyR1), cysteine 3635 (C3635) is viewed as dominantly responsible for the redox regulation of the channel. Here, we showed that the corresponding cysteine 3602 (C3602) in hRyR2 does not participate in intersubunit cross-linking and plays a limited role in the hRyR2 regulation by CaM during oxidative stress. Collectively, these results suggest that redox-mediated intersubunit cross-linking is an important regulator of hRyR2 function under pathological conditions associated with oxidative stress.

Original languageEnglish (US)
Article number101729
JournalRedox Biology
Volume37
DOIs
StatePublished - Oct 2020

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health Grants HL130231 (to A.V.Z.), HL092097 and HL138539 (to R.L.C), and AG026160 (to D.D.T). This study was also supported by a James DePauw pilot grant, Loyola University Chicago (to A.V.Z.). The authors would like to thank Dr. Christopher George (University of Cardiff, UK) for providing the vector encoding the GFP-hRyR2 and Dr. Ino for donating the R-CEPIA1er vector.

Funding Information:
This work was supported by the National Institutes of Health Grants HL130231 (to A.V.Z.), HL092097 and HL138539 (to R.L.C), and AG026160 (to D.D.T). This study was also supported by a James DePauw pilot grant, Loyola University Chicago (to A.V.Z.). The authors would like to thank Dr. Christopher George (University of Cardiff, UK) for providing the vector encoding the GFP-hRyR2 and Dr. Ino for donating the R-CEPIA1er vector.

Publisher Copyright:
© 2020 The Authors

Keywords

  • Ca signaling
  • Ca waves
  • Ryanodine receptor
  • Sarcoplasmic reticulum and oxidative stress

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

Fingerprint Dive into the research topics of 'The functional significance of redox-mediated intersubunit cross-linking in regulation of human type 2 ryanodine receptor'. Together they form a unique fingerprint.

Cite this