1. Supplemental Data for Two rings of negative charges in the cytosolic vestibule of type-1 ryanodine receptor modulate ion fluxes. by Le Xu, Ying Wang, Dirk Gillespie, and Gerhard Meissner.

2. Fig. 1S. Western Blot analysis of protein expression of WT-RyR1 and RyR1 mutants in HEK 293 cells. Crude membrane fractions (20 µg protein) were loaded on 3-12% SDS polyacrylamide gels. After overnight transfer to polyvinyl pyrrolidine fluoride membrane, the membrane was blocked with 5 % milk and 0.1% Tween-20 and exposed to a monoclonal antibody raised against RyR1 (mAb RyRD110) and horseradish peroxidase-conjugated antimouse antibody.

3. Fig. 2S. Caffeine-induced Ca 2+ release in HEK 293 cells expressing RyR1-D4945N, -E4948Q, -E4952Q and E4955Q. HEK 293 cells were transfected with indicated RyR1 mutant cDNAs. The fluorescence intensity of Fluo-4 loaded cells was measured before and after addition of 10 mM caffeine. Arrows indicate the addition of caffeine. Representative traces are shown.

4. Fig. 3S. Ca 2+ dependence of [ 3 H]ryanodine binding to WT-RyR1 and RyR1 mutants. Specific [ 3 H]ryanodine binding for cells transfected with WT-RyR1(●), RyR1-D4938N(○), RyR1-D4945N(▼), E4952Q (  ) and E4955Q ( ■ ) was determined as described in Experimental Procedures in 250mM KCl, 150 mM sucrose, 20 mM imidazole, pH 7.0, and 5 mM glutathione (oxidized) media containing 2.5nM [ 3 H]ryanodine and the indicated concentrations of free Ca 2+. Data are mean of four to six experiments  S.E.

5. Fig. 4S. Effects of cytosolic Ca 2+ on RyR1-D4938N channel activity. Single channel currents were recorded at -35 mV in symmetrical 250 mM KCl at indicated cytosolic Ca 2+ concentration as downward inflections from the closed state (c--). WT-RyR1 and RyR1-D4945N and -E4955Q showed a similar Ca 2+ -dependence of single channel activities.