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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
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-20 0 20 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 -1 log [Ca 2+ ] cyt Caffeine Caffeine igure 1 1 µM ts here….These are the kind of results published in the typical non-physiological bilayer c P). I think Caff = 5 mM here. 50 ms 5 pA

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Page 1: -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

-20 0 20

Vm, mV

10 nM

100 nM

1 mM

[Ca2+]cyt

Control

100 nM

1 µM

5 µM

100 µM

2 mM 50 mM

Control

Ope

n P

roba

bilit

y (P

o)

0.6

0.4

0.2

0.0

0.8

1.0

-7 -6 -5 -4-8 -9 -3 -2 -1

log [Ca2+]cyt

Caffeine

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

Page 2: -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

Figure 2

Ope

n P

roba

bilit

y1 µM

5Caffeine, mM

Ope

n P

roba

bilit

y

0.6

0.8

1.0

0 2010

0.4

0.2

0.015

A.

100 nM

5 nM

~10 µM

1 mM

50 mM

B.

0.6

0.8

1.0

0.4

0.2

0.05

Caffeine, mM0 2010 15

Variable cytosolic Ca2+

at 50 mM luminal Ca2+

Variable lumenal Ca2+

at 100 nM cytosolic Ca2+

Here We Defined Action of Cytosolic & Lumenal CaSimple solutions = no cyto Mg-ATP

Could include Scatchard-likePlot. Showing Km changes

But Vmax does not.

Could include Scatchard-likePlot. Showing Vmax changes

But Km similar.

Page 3: -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

1.0 2.00.5 0

Mg2+, mM1.5

ATP, mM3 51 0 2 4

A.

B.

100 nM Ca2+

cytosolic50 mM Ca2+ luminal

2 mM Caffeine

100 nM Ca2+

cytosolic50 mM Ca2+ 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

Page 4: -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

-7

Ope

n P

roba

bilit

y

0.6

0.8

1.0

-8 -4-6

0.4

0.2

0.0

-5

0 (Control)

20 mM

5 mM

log [Ca2+ ]cyt

[Caffeine ]

Combined action of Mg-ATP and CaffeineLines = 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

Page 5: -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

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

01

20 mM Caffeine

1 mM Caffeine

0

1

0

1

0

1

5 mM Caffeine

50 ms

5 p

A

0

1

Control

5 100 15 20[Caffeine] (mM)

1 20 3[Caffeine] (mM)

Ope

n P

roba

bilit

y

0.4

0.3

0.2

0.1

0

Ope

n P

roba

bilit

y 10-2

10-3

10-4

A. B.

2x

3x

5x Rest Po

Page 6: -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

Figure 6

[Caffeine] (mM)

Mea

n O

pen

Tim

e (

ms)

A. B.

5 100 15[Caffeine] (mM)

20

[Caffeine] (mM)

Mea

n O

pen

Tim

e (

ms)

100

10O

pen

Eve

nt F

requ

ency

(s-1

)

Ope

n E

vent

Fre

quen

cy (

s-1) 10

1

0.1

0.015 100 15[Caffeine] (mM)

20

1 20 3

40

10 2x

3x

5x Rest MOT4x

1 20 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)

Page 7: -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

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 testingthe predictions above would be great.

Problem is that if predictions are wrong then wewould have to explain why.


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