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Published bySibyl Lambert Modified over 2 years ago

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-20020 Vm, mV 10 nM 100 nM 1 mM [Ca 2+ ] cyt Control 100 nM 1 µM 5 µM 100 µM 2 mM 50 mM Control Open Probability (Po) 0.6 0.4 0.2 0.0 0.8 1.0 -7-6-5-4-8 -9 -3-2 log [Ca 2+ ] cyt Caffeine Figure 1 1 µM Story starts here….These are the kind of results published in the typical non-physiological bilayer conditions (no cyto Mg-ATP). I think Caff = 5 mM here. 50 ms 5 pA

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Figure 2 Open Probability 1 µM 5 Caffeine, mM Open Probability 0.6 0.8 1.0 02010 0.4 0.2 0.0 15 A. 100 nM 5 nM ~10 µM 1 mM 50 mM B. 0.6 0.8 1.0 0.4 0.2 0.0 5 Caffeine, mM 0201015 Variable cytosolic Ca 2+ at 50 mM luminal Ca 2+ Variable lumenal Ca 2+ at 100 nM cytosolic Ca 2+ Here We Defined Action of Cytosolic & Lumenal Ca Simple solutions = no cyto Mg-ATP Could include Scatchard-like Plot. Showing Km changes But Vmax does not. Could include Scatchard-like Plot. Showing Vmax changes But Km similar.

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1.02.00.5 0 Mg 2+, mM 1.5 ATP, mM 351 024 A. B. 100 nM Ca 2+ cytosolic 50 mM Ca 2+ luminal 2 mM Caffeine 100 nM Ca 2+ cytosolic 50 mM Ca 2+ luminal 10 mM Caffeine Here ATP EC50 Defined at 2 mM Caff Here Mg IC50 Defined at 10 mM Caff Now, we start adding Cytosolic ATP and Mg Figure 3

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-7 Open Probability 0.6 0.8 1.0 -8-4-6 0.4 0.2 0.0 -5 0 (Control) 20 mM 5 mM log [Ca 2+ ] cyt [Caffeine ] Combined action of Mg-ATP and Caffeine Lines = data from figure 1 (simple solutions no ATP-Mg before/after caff) Points are as labeled but now with ATP-Mg present. (net inhibition induced by cyto Mg-ATP is overcome by Caff addition ) Figure 4

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Now the GUTS…. Caffeine action on Po at in resting quasi-physiological solutions. (100 nM Ca cyto, high Ca lum, cyto Mg-ATP present) (2x rest Po occurs with 0.47 mM Caff, 3x at 0.87 mM, 4x at 1.11mM & 5x at 1.27 mM) Figure 5 0 1 0 1 0 1 0 1 20 mM Caffeine 1 mM Caffeine 0 1 0 1 0 1 5 mM Caffeine 50 ms 5 pA 0 1 Control 510 0 1520 [Caffeine] (mM) 12 0 3 Open Probability 0.4 0.3 0.2 0.1 0 Open Probability 10 -2 10 -3 10 -4 A. B. 2x 3x 5x Rest Po

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Figure 6 [Caffeine] (mM) Mean Open Time (ms) A. B. 510015 [Caffeine] (mM) 20 [Caffeine] (mM) Mean Open Time (ms) 100 10 Open Event Frequency (s -1 ) 10 1 0.1 0.01 510015 [Caffeine] (mM) 20 1203 40 10 2x 3x 5x Rest MOT 4x 12 0 3 0.6 0.1 2x 3x 5x Rest Freq. More GUTS…. Caffeine action on event freq and mean open time (MOT) in resting quasi-physiological solutions. (2x freq at 0.85 mM Caff, 3x at 1.27 mM, 4x at 1.7mM & 5x at 2.1 mM) (2x MOT at 0.42 mM Caff, 3x at 1.76 mM, 4x at 1.0mM & 5x at 1.3 mM)

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Figure 7 Make some RyR2 Function Predictions Like…. X mM Caff doubles MOT at rest conditions X mM Caff doubles Event Frequency at rest conditions If Possible, Sparks measurements testing the predictions above would be great. Problem is that if predictions are wrong then we would have to explain why.

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