RyR1-related myopathy mutations in ATP and calcium binding sites impair channel regulation

Fig. 1. Architechture of RyR1 channel. A Schematic diagram of the domain architecture of RyR1. B Coulombic density map of RyR1 (PDBID: 7M6A). The accessory protein calstabin-1 is shown in yellow. Zoom in showing Calcium (C), ATP (D), and caffeine (E) binding sites with the coordinating residues [14, 76]

Fig. 2. Structure of the ATP binding site. A The adenine ring binding site (T4979) and triphosphate tail interacting residues (K4211, K4214, and R4215) are labeled and depicted in stick representation. B [α-32P]-ATP/[3H]-ryanodine binding to ER microsomes of HEK293 cells expressing WT RyR1, T4979F, and K4211S/K4214S/R4215S. C, [3H]-ryanodine binding to ER microsomes of HEK293 cells expressing WT RyR1 and ATP binding site mutants in response to ATP. Data are presented as the mean ± S.E.M. N = 3 for each group. **P < 0.01

Fig. 3. Effects of ATP binding site mutations on the gating of RyR1 channels. A Representative single channel traces of WT RyR1 (top), T4979F (middle), and K4211S/K4214S/R4215S (bottom) under 10 µM Ca2+ only (left) or 10 µM Ca2+ and 1 mM ATP (right). B Po, C To, and D Tc of single WT RyR1, T4979F, and K4211S/K4214S/R4215S mutants. Data are presented as mean ± S.E.M from 6 single channels for each group. *P < 0.05

Fig. 4. Structure of the calcium binding site. A ribbon structure of the Ca2+ binding site showing interacting residues. B [3H]-ryanodine binding to ER microsomes of HEK293 cells expressing WT RyR1 and calcium binding site mutants. Data are presented as mean ± S.E.M from 6 single channels for each group. **P < 0.01

Fig. 5. Effects of mutations in the Ca2+ binding site on the gating of RyR1 channels. Representative single channel traces of WT RyR1 (A), E3893A (B), E3893D (C), E3967A (D), and E3967D (E), under 150 nM Ca2+, 10 µM Ca2+, or 10 mM Ca2+. F, Po versus Ca2+ concentration curve. Data are presented as mean ± S.E.M from 6 single channels for each group

Fig. 6. ATP binding and single channel analyses of the RyR1-RM patient-related ATP binding site mutants. A [α-32P]-ATP/ [3H]-ryanodine binding to ER microsomes of HEK293 cells expressing WT RyR1, T4979M, A538T and A538T/T4979M. B Single channel analysis of WT RyR1, T4979M, A538T and A538T/T4979M (bottom) under 10 µM Ca2+ only (left) or 10 µM Ca2+ and 1 mM ATP (right). C [3H]-ryanodine binding to ER microsomes of HEK293 cells expressing WT RyR1, T4979M, A538T and A538T/T4979M. Data are presented as mean ± S.E.M from 6 for each group

Fig. 7. Single channel analysis and [3H]-binding of the RyR1-RM patient-related Ca2+ binding site mutants. A Representative single channel traces of WT RyR1 and Q3970K under 150 nM Ca2 + , 10 µM Ca2+, or 10 mM Ca2+. B Po versus Ca2+ concentration curve. C [3H]-ryanodine binding to ER microsomes of HEK293 cells expressing WT RyR1 and Q3970K mutant. Data are presented as mean ± S.E.M from 6 single channels for each group