Article Figures & Data
Figures
- Fig. 1.
Dantrolene delays the onset of agonist-induced Ca2+ release from SR vesicles. Extravesicular Ca2+ level was monitored using APIII. SR vesicles were loaded with four subsequent aliquots of Ca2+ using SERCA activity. Injections of Ca2+ are labeled with asterisks (*). (A) Ca2+ release was triggered using 4CMC in 1 or 3 mM Mg2+ under control conditions (curves “a” and “b”) or in the presence of 10 μM dantrolene (curves “ad” and “bd”) (n = 3 to 4). (B) Ca2+ release curves recorded in 0.5 or 1.5 mM ATP in the absence (curves “a” and “A,” respectively) or presence of dantrolene (curves “ad” and “Ad”). Addition of dantrolene or DMSO is labeled with white and the addition of 4CMC with black arrowheads. (C) Spontaneous Ca2+ leak from SR vesicles after thapsigargin-treatment. (D) Transmittance-curve of untreated vesicles in the presence of 1 mM Mg2+ and 0.5 mM ATP. At the end of measurements shown in panels C and D, Ca2+ release was elicited using 4CMC to verify the functional integrity of the vesicles.
- Fig. 2.
Dantrolene suppresses RyR Po in lipid bilayers. (A and B) Single-channel currents of RyR1. Openings of the channel correspond to downward deflections. Representative current traces of a single channel under control conditions (50 μM Ca2+), after the addition of 1 mM Mg2+ and 0.5 mM ATP, are shown, respectively. Bottom trace: channel current in the presence of 1 mM Mg2+, 0.5 mM ATP, and 10 μM dantrolene. Average Po are shown above the representative current curves. Quantitative data are presented in (C). Data are expressed as mean ± S.D. Individual data points are represented by small dots.
- Fig. 3.
Dantrolene remained ineffective in the presence of calmodulin. (A) Western blot analysis of CaM in SR microsomes and a purified RyR sample. Whole-cell homogenate and purified CaM served as positive control. (B) Single-channel currents recorded in 100 nM Ca2+, 1 mM ATP, and 100 nM CaM under control conditions (top traces) and during dantrolene treatment (bottom trace). (C) Po values of six individual channels under control conditions and during dantrolene treatment.
- Fig. 4.
Representation of the putative DBS and its interdomain interactions in the 3D structure of RyR1. (A) Two subunits (caffeine/ATP/Ca2+ dataset, 5TAQ.pdb) of RyR1 are shown by cartoon representation (side view). ATP, Ca2+, and caffeine are shown by sphere representation and are colored by magenta and orange, respectively. NSol domain (393–627) is shown by cyan. The DBS (590–609) is highlighted by red. The DP4 region (2442–2477) is shown by orange. Sequence is numbered according to the rabbit Ryr1. (B) Shows the putative interaction surfaces in the proximity of the DBS. The NSol domain (393–627) is highlighted by cyan, the SP1a/RyR domain 3 (SPRY3) domain (1242–1656) by green, the junctional solenoid (JSol) domain (1657–2144) by orange, and the bridging solenoid (BSol) domain (2145–3613) by violet. DBS (590–609) is highlighted by red. Regions of neighboring domains located in the proximity of DBS are circled. Hydrogen bonds are formed between aa N1678-Q618, G1677-R615, D1658-H597; Q2169-A613; and D591-R1594, D591-R1594, L590-R1594. Well-known mutation sites around the DBS are shown by blue sticks and are indicated by blue numbers and arrows. R614 of human Ryr1 corresponds to R615 in rabbit Ryr1. See text for further details.
Additional Files
Data Supplement
- Supplemental Figure -
Supplementary Figure 1 - Dantrolene is ineffective on cardiac sarcoplasmic reticulum vesicles.
- Supplemental Figure -
