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 hRyR2^WT and hRyR2^C2991S. b Summary of the effect of the increasing diamide concentrations on the level of RyR2 monomeric form for hRyR2^WT and hRyR2^C2991S 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 hRyR2^WT (black) and hRyR2^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 hRyR2^WT (black) or hRyR2^C2991S (red). Data are shown as means ± SE (25 μM diamide: n = 29 for hRyR2^WT and 28 cells for hRyR2^C2991S; 50 μM diamide: n = 40 for hRyR2^WT and 48 cells for hRyR2^C2991S) and were analyzed using a two-sided unpaired t-test.