-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...
TRANSCRIPT
-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
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.
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
-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
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
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)
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.