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

Fig. 1. Cell model for functional study of redox-sensitive cysteine residues in hRyR2. a Representative confocal image of the HEK293 cell model for the functional studies of redox-sensitive cysteines in RyR2. Corresponding images demonstrate the cells under transmitted light and the expression of GFP-tagged hRyR2 and R-CEPIA1er [Ca2+]ER sensor. The images of co-localized GFP-tagged RyR2 and R-CEPIA1er were obtained for corresponding HEK293 cells in all confocal experiments mentioned in this paper. b Representative recordings of Ca2+ waves generated by hRyR2 in HEK293 cells. Diamide was added to induce hRyR2 oxidation and cross-linking. Caffeine (Caf; 10 mM) and ionomycin (2 µM) in 10 mM Ca2+ were used to normalize the signal. The dashed line indicates the level of ER Ca2+ load. c Western blot of the disulfide cross-linking of hRyR2 caused by increasing concentrations of diamide. DTT (100 mM) was added to the samples to reverse hRyR2 oxidation and cross-linking (data representative of 3 independent experiments).

Fig. 2. Localization of the N-terminal domains A and B in RyR2. a 3D visualization of the human RyR2 cryo-EM model (PDB ID: 7UA5) is shown in grey. Domains A and B are shown in blue and red, respectively. Cysteine residues are shown in green. b Localization of the truncation site in the AB-truncated hRyR2 mutant (hRyR2ΔAB) on the primary structure.

Fig. 3. Truncation of the N-terminal domains A and B in RyR2 inhibits intersubunit disulfide cross-linking and diamide-induced depletion in [Ca2+]ER. a Representative western blot image of the hRyR2 cross-linking caused by diamide in HEK293 cells expressing GFP-hRyR2 and hRyR2ΔAB. All representative western blot images are presented as a top part, above the MW of 250 kDa, including the loading wells area. b Summary of the effect of the increasing diamide concentrations on the level of RyR2 monomeric form for hRyR2WT and hRyR2ΔAB channels expressed in HEK293 cells. Data are shown as means ± SE (n = 4, each n represents data from an independent experiment) and were analyzed using a two-sided unpaired t-test. c Representative recordings of Ca2+ waves from HEK293 cells expressing hRyR2WT (black) and hRyR2ΔAB (red) in control condition followed by the application of 50 µM diamide. The dashed lines indicate the level of ER Ca2+ load. Caffeine (Caf; 10 mM) and ionomycin (2 µM) in 10 mM Ca2+ were used to normalize the signal. d Summary of the effect of 50 µM diamide on ER Ca2+ load in HEK293 cells transfected with hRyR2WT (black) or hRyR2ΔAB (red). Data are shown as means ± SE (n = 22 for…

Fig. 4. N-terminal domain A of hRyR2 has an allosteric effect on cross-linking. a Representative western blot image of hRyR2 cross-linking caused by diamide in HEK293 cells expressing hRyR2WT and hRyR2ΔA. b Summary of the effect of the increasing diamide concentrations on the level of RyR2 monomeric form for hRyR2WT and hRyR2ΔA channels expressed in HEK293 cells. Data are shown as means ± SE (n = 5, each n represents data from an independent experiment) and were analyzed using a two-sided unpaired t-test.

Fig. 5. Cysteines 1078 and 2991 face one another at the neighboring subunits of hRyR2 and are accessible for oxidation depending on channel conformation. a Suggested localization of cysteines 1078 and 2991 in the 3D cryo-EM model of the closed hRyR2 (PDB ID: 7UA5). Helical domain 2 is shown in cyan. P1 and P2 domains are shown in magenta and orange, respectively. SPRY1 and SPRY2 domains are shown in slate and marine, respectively. The cross-linking cysteines 1078 and 2991 are shown in green. The right panel illustrates the local environments of C1078 and C2991 in closed (PDB ID: 7UA5) and open (PDB ID: 7UA9) conformations. The number in the square brackets represents the cysteine thiol group’s solvent-accessible surface area (ASA) in Å2. Black dashed lines between residue side chains represent the van der Waals interactions, and green dashed lines represent hydrogen bonds. b Localization of cysteines 1078 and 2991 on the primary hRyR2 structure and a potential site of the disulfide formation caused by oxidative stress.

Fig. 6. Cysteine 1078 directly participates in hRyR2 cross-linking and functional activation by diamide. a Representative western blot image of hRyR2 cross-linking caused by diamide in HEK293 cells expressing hRyR2WT and hRyR2C1078S. b Summary of the effect of the increasing diamide concentrations on the level of RyR2 monomeric form for hRyR2WT and hRyR2C1078S channels expressed in HEK293 cells. Data are shown as means ± SE (n = 5, each n represents data from an independent experiment) and were analyzed using a two-sided unpaired t-test. c Representative recordings of Ca2+ waves from Flp-In T-Rex-293 SERCA2a stable line cells expressing hRyR2WT (black) and hRyR2C1078S (red) in control condition followed by the application of 25 µM diamide. Caffeine (Caf; 10 mM) and ionomycin (2 µM) in 10 mM Ca2+ were used to normalize the signal. d Summary of the effect of 25 and 50 µM diamide on ER Ca2+ load in cells transfected with hRyR2WT (black) or hRyR2C1078S (red). Data are shown as means ± SE (25 μM diamide: n = 43 for hRyR2WT and 40 cells for hRyR2C1078S; 50 μM diamide: n = 27 for hRyR2WT and 36 cells for hRyR2C1078S) and were analyzed using a two-sided unpaired t-test.

Fig. 7. Cysteine 2991 directly participates in hRyR2 cross-linking and functional activation by diamide. a Representative western blot image of hRyR2 cross-linking caused by diamide in HEK293 cells expressing hRyR2WT and hRyR2C2991S. b Summary of the effect of the increasing diamide concentrations on the level of RyR2 monomeric form for hRyR2WT and hRyR2C2991S channels expressed in HEK293 cells. Data are shown as means ± SE (n = 3, each n represents data from an independent experiment) and were analyzed using a two-sided unpaired t-test. c Representative recordings of Ca2+ waves from Flp-In T-Rex-293 SERCA2a stable line cells expressing hRyR2WT (black) and hRyR2C2991S (red) in control condition followed by the application of 25 µM diamide. Caffeine (Caf; 10 mM) and ionomycin (2 µM) in 10 mM Ca2+ were used to normalize the signal. d Summary of the effect of 25 and 50 µM diamide on ER Ca2+ load in cells transfected with hRyR2WT (black) or hRyR2C2991S (red). Data are shown as means ± SE (25 μM diamide: n = 29 for hRyR2WT and 28 cells for hRyR2C2991S; 50 μM diamide: n = 40 for hRyR2WT and 48 cells for hRyR2C2991S) and were analyzed using a two-sided unpaired t-test.

Fig. 8. Cysteines 1078 and 2991 promote hRyR2 activation during oxidative stress by forming a disulfide bond. a Representative western blot image of hRyR2 cross-linking caused by diamide in HEK293 cells expressing hRyR2WT and hRyR2C1078S/C2991S. b Summary of the effect of the increasing diamide concentrations on the level of RyR2 monomeric form for hRyR2WT and hRyR2C2991S channels expressed in HEK293 cells. Data are shown as means ± SE (n = 5, each n represents data from an independent experiment) and were analyzed using a two-sample t-test. c Representative recordings of Ca2+ waves from Flp-In T-Rex-293 SERCA2a stable line cells expressing hRyR2WT (black) and hRyR2C1078S/C2991S (red) in control condition followed by the application of 25 µM diamide. Caffeine (Caf; 10 mM) and ionomycin (2 µM) in 10 mM Ca2+ were used to normalize the signal. d Summary of the effect of 25 and 50 µM diamide on ER Ca2+ load in cells transfected with hRyR2WT (black) or hRyR2C1078S/C2991S (red). Data are shown as means ± SE (25 μM diamide: n = 39 for hRyR2WT and 44 cells for hRyR2C1078S/C2991S; 50 μM diamide: n = 24 for hRyR2WT and 27 cells for hRyR2C1078S/C2991S) and were analyzed using a two-sided unpaired…