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

• Published in: Redox biology Vol. 37; p. 101729
• Main Authors: Nikolaienko, Roman, Bovo, Elisa, Rebbeck, Robyn T., Kahn, Daniel, Thomas, David D., Cornea, Razvan L., Zima, Aleksey V.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2020; Elsevier
• Subjects: Animals; Ca2+ signaling; Ca2+ waves; Calcium - metabolism; Calcium Signaling; HEK293 Cells; Humans; Oxidation-Reduction; Rabbits; Research Paper; Ryanodine receptor; Ryanodine Receptor Calcium Release Channel - genetics; Ryanodine Receptor Calcium Release Channel - metabolism; Sarcoplasmic reticulum and oxidative stress; Ca2+ waves; Sarcoplasmic reticulum and oxidative stress; Ca2+ signaling; Ryanodine receptor; Ca(2+) waves; Ca(2+) signaling
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2020.101729

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. [Display omitted] •Oxidative stress promotes cardiac ryanodine receptor (RyR2) intersubunit crosslinking.•Human RyR2 crosslinking promotes Ca leak and calmodulin dissociation.•RyR2 C3602 is not involved in crosslinking, slightly affects calmodulin binding.•RyR2 crosslinking is an important pathology related RyR2 regulator.