Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State

Figures

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  Figure 1.. DPc10 effect on CaM/RyR2 binding. (A) RyR2 in SR isolated from porcine heart was decorated with D-FKBP, then treated with (solid symbols) or without (open symbols) DPc10 (50 μM) for 60 min (25°C). The concentration dependence of A-CaM binding to RyR2 was measured using FRET after further incubating with A-CaM at the indicated concentrations (150 min, 25°C), in 30 nM or 300 μM Ca2+, as indicated. (B), Parameters Kd and Emax obtained from fitting the data shown in A to a single rectangular hyperbola. Data represent the mean ± SE from 4 paired experiments. *significantly different from no-DPc10 controls; #significantly different from the 30 nM Ca2+ condition, as determined by p ≤ 0.05 (unpaired Student’s t-tests).

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  Figure 2.. FLT-FRET detection of DPc10 structural effect on CaM/RyR2 complex. (A) RyR2 in cardiac SR membranes was decorated with D-FKBP, then treated with (solid line) or without (dotted line) DPc10 (50 μM), and finally incubated with saturating (0.8 μM) A-CaM. A representative FLT-FRET experiment is illustrated by fluorescence decays of D-FKBP without A-CaM (grey, D) and with (black, DA) A-CaM. (B) Multi-exponential analysis of panel A FLT-FRET decays resolves a one-distance Gaussian distribution of the separation between D-FKBP and A-CaM on RyR2 in 30 nM (blue) or 30 μM (red) Ca2+. Distributions are centered at the distances indicated in panel A, and their widths (FWHMs) shown in panel C. (D) Effect of Ca2+ and DPc10 on the mole-fraction of D-FKBP not participating in FRET (XD-only). Data shown as means ± SE; n=4-6. *p≤0.5 (paired Student’s t-test).

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  Figure 3.. CaM influence on DPc10/RyR2 binding. RyR2 in cardiac SR membranes was decorated with D-FKBP, and then pre-incubated 40 min with or without 30 μM CaM. These samples were then further incubated (40 min) with a series of [A-DPc10], as indicated, at 30 nM Ca2+ (A) or 300 μM Ca2+ (B). DPc10/RyR2 binding was measured using FRET from D-FKBP to A-DPc10. Fitting was performed using the Hill function; fitting parameters are shown in Table1. Data shown as mean ± SE, n = 4-6 *p ≤ 0.05 (paired Student’s t-test).

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  Figure 4.. RyR2 in cardiac SR membranes were decorated with either D-14-FKBP, D-32-FKBP, D-49-FKBP, D-85-FKBP, and incubated with A-DPc10. Fitting parameters are shown in Table 2 (n=4-6).

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  Figure 5.. Location of the A-DPc10 biosensor within the RyR2 structure. The RyR2 cryo-EM structure with CaM and FKBP bound (PDB ID: 6JI8) [28] is shown as ribbon representation. Two RyR2 protomers are shown in green and the other two are shown in gray to emphasize protomer boundaries. CaM is shown in purple and FKBP is shown in blue. The helices of HD1 of RyR2 that correspond to the sequence range that the DPc10 peptide is derived from (2460-2495) are shown as red cylinders. Inserted in this location is A-DPc10 shown as a yellow cylinder with the fluorophore shown as a yellow star. The fluorophore is located in the cavity below HD1, NTDA, and NTDC, between the Central domain of one protomer and the SPRY2 domain of the adjacent protomer. Structure figures were created using VMD [34].