![Verification of the B–A–C–A–C network.(A) Caffeine-induced changes in [Ca2+]cyt determined by fura-2 in the WT and mutants (E40A and D61A). Caffeine was applied at the time points indicated by the black horizontal bar. (B and C) Comparison of the peaks of [Ca2+]cyt transiently induced by 0.3 mM (B; n = 161–233) and 10 mM (C; n = 161–245) caffeine in the WT (black) or mutants (gray). (D) Comparison of resting [Ca2+]cyt in the WT (black) and mutants (gray; n = 50–93). (E) Structural deviations observed in the MD simulations. Plots of the RMSD of the Cα atoms from the initial structures of NTD-RYR1s (E40A and D61A) as a function of time are shown. Calculations of each RMSD were performed using MD trajectories from the representative results. The trajectory during 20–50 ns (gray horizontal bar) was analyzed for hydrogen bonds/salt bridges (see F and G). (F and G) Analyses of probabilities of forming hydrogen bonds/salt bridges during the MD simulations. Hydrogen bonds/salt bridges between AC and BC domains are considerably strong. However, hydrogen bonds/salt bridges between AB domains are relatively weak. (H and I) Superimposition of the monomer of the WT (yellow) and of the E40A (H) or D61A (I) mutant after 50 ns of the MD simulation. The result shown here is a representative of 20 calculations. The BC domains rotated 5.7 degrees or 14.9 degrees with respect to the A domain in the E40A (H) or D61A (I) mutants, respectively. The average rotation angle is shown in Fig. S3 E. The rotation axis is indicated by a black line. (J and K) Closeup view around residue 402 is the same as shown in Fig. 3. Colors of domains are the same as shown in Fig. 1. Dashed lines represent hydrogen bonds/salt bridges. The same view after 50 ns of the MD simulation of the E40A (J) or D61A (K) mutant is shown. Although E40A cannot form hydrogen bonds/salt bridges between R402 and S406 via E40, interaction between AB domains via D61 remains (J). However, in D61A, the interaction between AC domains via E40 remains, but there is no AB interaction. Values are shown as mean ± SEM. *P < 0.0001 by Student's t test compared with the WT.](https://cdn.rupress.org/rup/content_public/journal/jgp/152/1/10.1085_jgp.201812235/5/jgp_201812235_fig5.png?Expires=1767755446&Signature=fq2IGxoq1JBeKVOHupQ9-pSWgzu0fS4Ix0FrAsH8funXpLTHbt87hxRmhD2bamzNiKjjsk648JZluOV2K9gOE3vItsq4EHMq0miKb1OYG1AaANFa9tzoiWGHQo6zF4SvqKnZ84OOS8vSbqy-P9V8~Q8De8pA41OH3wcirFoF3OnkUFVPlJHF7M7k5Yx0Ukr~xGdpn3RACPRNCE-XKXOpwBy6rppKjpkaDF4JyfyLccMuaMkHSzimBjO-~oVl1ZBQbhvxHySG4BDNJyXfL33DRr6~eFFvrTuqI5t8CDLh8--~pTyzwU9vSfOKoDDdkt3TiqnSq8XtfSDw8rbFLBL-AQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Verification of the B–A–C–A–C network. (A) Caffeine-induced changes in [Ca2+]cyt determined by fura-2 in the WT and mutants (E40A and D61A). Caffeine was applied at the time points indicated by the black horizontal bar. (B and C) Comparison of the peaks of [Ca2+]cyt transiently induced by 0.3 mM (B; n = 161–233) and 10 mM (C; n = 161–245) caffeine in the WT (black) or mutants (gray). (D) Comparison of resting [Ca2+]cyt in the WT (black) and mutants (gray; n = 50–93). (E) Structural deviations observed in the MD simulations. Plots of the RMSD of the Cα atoms from the initial structures of NTD-RYR1s (E40A and D61A) as a function of time are shown. Calculations of each RMSD were performed using MD trajectories from the representative results. The trajectory during 20–50 ns (gray horizontal bar) was analyzed for hydrogen bonds/salt bridges (see F and G). (F and G) Analyses of probabilities of forming hydrogen bonds/salt bridges during the MD simulations. Hydrogen bonds/salt bridges between AC and BC domains are considerably strong. However, hydrogen bonds/salt bridges between AB domains are relatively weak. (H and I) Superimposition of the monomer of the WT (yellow) and of the E40A (H) or D61A (I) mutant after 50 ns of the MD simulation. The result shown here is a representative of 20 calculations. The BC domains rotated 5.7 degrees or 14.9 degrees with respect to the A domain in the E40A (H) or D61A (I) mutants, respectively. The average rotation angle is shown in Fig. S3 E. The rotation axis is indicated by a black line. (J and K) Closeup view around residue 402 is the same as shown in Fig. 3. Colors of domains are the same as shown in Fig. 1. Dashed lines represent hydrogen bonds/salt bridges. The same view after 50 ns of the MD simulation of the E40A (J) or D61A (K) mutant is shown. Although E40A cannot form hydrogen bonds/salt bridges between R402 and S406 via E40, interaction between AB domains via D61 remains (J). However, in D61A, the interaction between AC domains via E40 remains, but there is no AB interaction. Values are shown as mean ± SEM. *P < 0.0001 by Student's t test compared with the WT.