Solution change

1. [Ca2+]SR Rhod-2 Rhod-2 [Ca2+]SR Solution change Note: no great cytoplasmic release at peak of or rise in [Ca2+]SR. Therefore mitochondria cannot be loading cytoplasmic Ca2+. Also, it is obvious that high [Ca2+]SR areas are clearly transversal (SR terminal cisternae) and longitudinal areas (mitochodria) are low [Ca2+]. 081004a Effect of removing SO42- on SR [Ca2+] (SO42- is substituted for glutamate at the second data point in this figure). 081004a

2. 081004a_s132_4_7_rto.tif 081004a_s133_0_11_rto.tif Avg ratio series 133 #0 to 11 Avg ratio series132 #4 to 7 tv,255<rotate(100.*(rto0-0.4),flagrot)>0 B A Difference B-A, > 0 Difference, A-B, > 0

3. [Ca2+]SR Rhod-2 [Ca2+]SR Rhod-2 [Ca2+]SR Rhod-2 081004b Effect of removing SO42- on SR [Ca2+] (SO42- is substituted for glutamate at the second data point in this figure). [Ca2+]SR Rhod-2

4. Lots of mitochondria in mag-indo image—could be decreasing estimate of [Ca2+]. The SR emptied over the course of the next few minutes. Effect of removing SO42- on SR [Ca2+] (SO42- is substituted for glutamate at the second data point in this figure). 102804a

5. Rhod-2 [Ca2+]SR The fastest rise in [Ca2+]SR is perhaps around 500-600 μM s-1? Rhod-2 Rhod-2 [Ca2+]SR [Ca2+]SR 102804c Effect of removing SO42- on SR [Ca2+] (SO42- is substituted for 50mM EGTA at the second data point in this figure). Rhod-2 [Ca2+]SR

6. SO42- EGTA Effect of loading in 80 mM SO42-, 1 mM EGTA (100 nM Ca2+) and 0.4 mM Mg2+ for ~16 min, then switching to 50 mM EGTA (< 2 nM Ca2+) and 1 mM Mg2+. Total SR Ca is assayed at the end of the expt with caffeine. 30 mM caffeine caffeine 110904d

7. solution change 110804c Effect of removing SO42- on SR [Ca2+] (SO42- is substituted for 50mM EGTA at the 18-20 s time point in this figure).

8. Effect of removing 50 mM SO42- (isosmotically substituted with K+-Glut), 1 mM EGTA (100 nM Ca2+) and 0.4 mM Mg2+ on SR [Ca2+] (SO42- is substituted for 50mM EGTA). 50 mM SO42- EGTA 30 mMcaffeine XT’s here aswell 111604a

9. Effect of loading in 50 mM Pi, 1 mM EGTA (100 nM Ca2+) and 0.4 mM Mg2+ for ~17 min, then switching to 50 mM EGTA (< 2 nM Ca2+) and 1 mM Mg2+. Note: no sparks 110904a

10. Effect of loading in 50 mM Pi, 1 mM EGTA (100 nM Ca2+) and 0.4 mM Mg2+ for ~17 min, then switching to 50 mM EGTA (< 2 nM Ca2+) and 1 mM Mg2+. Note: no sparks 111604b Open pinhole

11. Effect of loading in 50 mM Pi, 1 mM EGTA (200 nM Ca2+) and 1 mM Mg2+ for ~17 min, then switching to 50 mM EGTA (< 2 nM Ca2+) and 1 mM Mg2+. Total SR Ca is assayed at the end of the expt with caffeine. 30 mM caffeine 110904c

12. Glut EGTA Effect of substituting K-Glut, 1 mM EGTA (100 nM Ca2+) and 0.4 mM Mg2+ [after 15 min loading] for 50 mM EGTA (< 2 nM Ca2+) and 1 mM Mg2+. 111004b

13. 50 μM TBQ SO42-, 100 nM Ca2+, 0.4 mM Mg2+ 50 mM EGTA, 1 mM Mg2+ 111804a

14. 50 μM TBQ SO42-, 100 nM Ca2+, 0.4 mM Mg2+ 50 mM EGTA, 1 mM Mg2+ 111804b

15. Saturated signal 30 mM caff 111604c