Leaky Ca²⁺ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice
J. Clin. Invest. Stephan E. Lehnart, et al. 118:2230 doi:10.1172/JCI35346 [Go to this article.]

Figure 3
Hippocampal Ryr2^RS/WT brain slices and channels exhibit burst activity, which can be inhibited by treatment with ryanodine or the RyR-stabilizing drug S107, respectively. (A and B) Continuous confocal Ca²⁺ fluorescence imaging of the Ryr2^RS/WT CA3 principal cell layer under control conditions (left), and seizure activity induced by low Mg²⁺ (0.5 mM) plus high K⁺ (8.5 mM) (middle) and following ryanodine (10 μM) treatment (right). Fluorescence (F) signals 1–3 in A correspond to regions of interest indicated by white circles in the CA3 layer in B. Data are representative of 3 experiments using Ryr2^RS/WT hippocampal slices; dimensions are as indicated. (C) H&E histology (left) and RyR2 immunohistochemistry (right) of the hippocampal CA3 region in Ryr2^RS/WT brain slices show increased RyR2 expression in the preserved principal cell layer. There were no histological abnormalities compared with WT (data not shown). (D) Representative Ryr2^RS/WT single-channel traces from vesicles of isolated hippocampus from sedentary mice (left), after injection of NE (5 mg/kg twice over 3 hours; middle) or after 1 week treatment with S107 (5 mg/kg/h) followed by NE treatment (5 mg/kg twice over 3 hours; right). P_o, mean open (To) and mean closed (Tc) times, closed state (c), and the fully open level (4 pA) are indicated. Thick bars above the 5-second traces indicate area shown at higher resolution in the 0.5-second traces. All-point histograms corresponding to the single-channel traces show increased numbers of partial openings (subconductance states) and overall increased activity of the brain channels from NE-treated Ryr2^RS/WT mice (middle histogram). The histogram on the right shows more channels in the closed state (0 pA), consistent with the channel-stabilizing properties of the drug S107 that enhances binding of the calstabin2 subunit to the channel. (E) Average P_o of WT and Ryr2^RS/WT brain channels under different treatment conditions as indicated. Single-channel measurements were performed at a cis (cytosolic) Ca²⁺ concentration of 150 nM. *P < 0.05 versus NE-untreated; ^†P < 0.05, NE-treated WT versus Ryr2^RS/WT. Each bar represents the average of 7–9 channels. Equivalent amounts of RyR2 were immunoprecipitated from brain homogenates with an RyR2 isoform–specific antibody followed by immunoblotting; bar graphs show the amount of PKA phosphorylation of RyR2 at Ser2808 (F) and the amount of calstabin2 bound to RyR2 (G) under the indicated conditions. Animals were treated with S107 via implantable osmotic pumps (5 mg/kg/h) for 7 days before NE stimulation. *P < 0.05 versus NE-untreated.