-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...
Post on 16-Dec-2015
227 Views
Preview:
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.
top related