Defective ryanodine receptor N-terminus inter-subunit interaction is a common mechanism in neuromuscular and cardiac disorders

Figures

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  FIGURE 1. MH mutation R164C disrupts RyR1 NTD tetramerization. Chemical cross-linking assays of HEK293 cell homogenates expressing NTRyR1−WT (rabbit RyR1 residues 1–915, cMyc-tagged) or the R164C mutant, NTRyR1-R164C. (A). Cell homogenates were incubated with glutaraldehyde for the indicated time points under reducing (10 mM DTT) conditions and analyzed by Western blotting using AbcMyc; monomer (M) and tetramer (T) are indicated with the arrows. (B). Densitometry analysis was carried out on the bands corresponding to tetramer and monomer moieties and used to calculate tetramer formation. Data (n = 7) are given as mean value ±SEM; statistical analysis was carried out using Mann-Whitney test.

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  FIGURE 2. Rescue of defective RyR2 NTD tetramerization in CPVT. (A). Peptide sequence of RyR1/2 β8-β9 (left) and β23-β24 loops (right). RyR1 and RyR2 pathogenic mutations are shaded and listed in Table 1. (B). Image of the RyR2 N-terminal inter-subunit interface generated using PyMol from the RyR2 closed state (5GO9) structure. The location of the CPVT mutation R169Q, as well as residues Q168 and D180 on one subunit that are in close apposition to residues M399 and D400 on the neighboring subunit, are depicted. The Q168-D400 and D180-M399 distances (in Å) are also indicated. (C). Chemical cross-linking assays of NTWT (human RyR2 residues 1–906, cMyc-tagged), NTQ168R, NTM399R, NTR169Q, NTR169Q+Q168R and NTR169Q+M399R expressed in HEK293 cells. Cell homogenates were incubated with glutaraldehyde for the indicated time points under reducing (10 mM DTT) conditions and analyzed by Western blotting using AbcMyc. Densitometry analysis was carried out on the bands corresponding to tetramer and monomer moieties and used to calculate tetramer formation. Data (n ≥ 7) are given as mean value ±SEM.