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Cysteines 1078 and 2991 cross-linking plays a critical role in redox regulation of cardiac ryanodine receptor (RyR).

ABSTRACT: The most common cardiac pathologies, such as myocardial infarction and heart failure, are associated with oxidative stress. Oxidation of the cardiac ryanodine receptor (RyR2) Ca²⁺ channel causes spontaneous oscillations of intracellular Ca²⁺, resulting in contractile dysfunction and arrhythmias. RyR2 oxidation promotes the formation of disulfide bonds between two cysteines on neighboring RyR2 subunits, known as intersubunit cross-linking. However, the large number of cysteines in RyR2 has been a major hurdle in identifying the specific cysteines involved in this pathology-linked post-translational modification of the channel. Through mutagenesis of human RyR2 and in-cell Ca²⁺ imaging, we identify that only two cysteines (out of 89) in each RyR2 subunit are responsible for half of the channel's functional response to oxidative stress. Our results identify cysteines 1078 and 2991 as a redox-sensitive pair that forms an intersubunit disulfide bond between neighboring RyR2 subunits during oxidative stress, resulting in a pathological "leaky" RyR2 Ca²⁺ channel.

SUBMITTER: Nikolaienko R

PROVIDER: S-EPMC10372021 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Cysteines 1078 and 2991 cross-linking plays a critical role in redox regulation of cardiac ryanodine receptor (RyR).

Nikolaienko Roman R Bovo Elisa E Kahn Daniel D Gracia Ryan R Jamrozik Thomas T Zima Aleksey V AV

Nature communications 20230726 1

The most common cardiac pathologies, such as myocardial infarction and heart failure, are associated with oxidative stress. Oxidation of the cardiac ryanodine receptor (RyR2) Ca<sup>2+</sup> channel causes spontaneous oscillations of intracellular Ca<sup>2+</sup>, resulting in contractile dysfunction and arrhythmias. RyR2 oxidation promotes the formation of disulfide bonds between two cysteines on neighboring RyR2 subunits, known as intersubunit cross-linking. However, the large number of cystei ...[more]

PMID: 37495581

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Project description: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.

Project description:Despite its importance and abundance of experimental data, the molecular mechanism of RyR2 activation by calcium is poorly understood. Recent experimental studies involving coexpression of wild-type (WT) RyR2 together with a RyR2 mutant deficient in calcium-dependent activation (Li, P., and S.R. Chen. 2001. J. Gen. Physiol. 118:33-44) revealed large variations of calcium sensitivity of the RyR tetramers with their monomer composition. Together with previous results on kinetics of Ca activation (Zahradníková, A., I. Zahradník, I. Györke, and S. Györke. 1999. J. Gen. Physiol. 114:787-798), these data represent benchmarks for construction and testing of RyR models that would reproduce RyR behavior and be structurally realistic as well. Here we present a theoretical study of the effects of RyR monomer substitution by a calcium-insensitive mutant on the calcium dependence of RyR activation. Three published models of tetrameric RyR channels were used either directly or after adaptation to provide allosteric regulation. Additionally, two alternative RyR models with Ca binding sites created jointly by the monomers were developed. The models were modified for description of channels composed of WT and mutant monomers. The parameters of the models were optimized to provide the best approximation of published experimental data. For reproducing the observed calcium dependence of RyR tetramers containing mutant monomers (a) single, independent Ca binding sites on each monomer were preferable to shared binding sites; (b) allosteric models were preferable to linear models; (c) in the WT channel, probability of opening to states containing a Ca2+-free monomer had to be extremely low; and (d) models with fully Ca-bound closed states, additional to those of an Monod-Wyman-Changeaux model, were preferable to models without such states. These results provide support for the concept that RyR activation is possible (albeit vanishingly small in WT channels) in the absence of Ca2+ binding. They also suggest further avenues toward understanding RyR gating.

Dataset Information

Cysteines 1078 and 2991 cross-linking plays a critical role in redox regulation of cardiac ryanodine receptor (RyR).

Publications

Cysteines 1078 and 2991 cross-linking plays a critical role in redox regulation of cardiac ryanodine receptor (RyR).

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