1. Reversible intra-SR precipitation of calcium, Ca sparks
and Ca-dependent inactivation
Brad
Jingsong
Gustavo
Leandro
Adom
Demetrio
Tom
Mike Stern
Julio Copello
Maura Porta
Mike Fill
Alma

2. SO42- widens frog sparks.
B and D are averages of events of 4ms < rt < 6 ms.
1022c914
10 mm
500 ms
3 F0 A C B
Frog, glutamate D
Frog, SO42-
10 ms D
2 mm

3. An interesting property of sparks, and its change by SO4
(more on this later)
reference
amplitude
SO4

4. SO4 increases spark width, rise time, while reducing amplitude
FWHM mm
rise time ms
frequency
s-1 (100 mm)-1
N, events
(cells)
Reference
0.88
0.06
1.53
0.07
5.55
0.41
5.3
2.5
1890
(12)
SO4
75 mM
0.63
0.03
2.02
0.02
7.31
0.32
3.3
1.3
359
(6)

5. SO4 induces two extreme classes of sparks,
distinguishable at high temporal resolution
5 mm
10 ms
2F0
“concerted”
“sequential”

6. An early hypothesis about mechanism
SO42-
Ca2+
CaSO4 P
Sarcoplasmic
Reticulum
Cytosol
Ksp = 36 mM2

7. Sulfate precipitates as Ca salt inside fractionated SR
(Demetrio and Tom, 2003)
sulfate
chloride

8. SEER and other studies of last three years

9. Shifted Excitation and Emission Ratioing of fluorescence
SEER
Shifted Excitation and Emission Ratioing of fluorescence
Launikonis et al.
J. Physiol. ‘05 1 2 3
High Ca
Low Ca
Excitation
Emission
l,nm
The technique we devised is named SEER. A description of it just went online in the J. Physiology two days ago.
SEER is a ratiometric technique, that takes advantage of the shift in both the excitation and the emission spectra that some dyes undergo when binding Ca.
By using both shifts, the sensitivity is greatly enhanced, and one can follow Ca transients inside small organelles, SR and mitochondria.
In the case of mag-indo-1 the emission ranges are at very short wavelengths, so that one can have an additional dye in the cytosol, and measure in parallel Ca in the SR and in the cytosol.
9 of 26

10. REFERENCE
SULFATE
WASHOUT A B C b c a

11. REFERENCE
PHOSPHATE
WASHOUT A B C b a c

12. Non-precipitating buffers do not change [Ca2+] in SR
(example, 40 mM citrate)

13. The source of Ca for the “surge” is the SR
SULFATE
50 EGTA
CAFFEINE
Ca SR
20 mm
Ca cytosol
100 nM Ca2+
0 Ca2+
SO42-
EGTA
caffeine
F/F0
R, [Ca2+]SR, mM
time, min a b c

14. final Ca level in sulfate is a function of final level in reference (glutamate)
[Ca2+]SR, mM
R in sulfate
R in glutamate, R0
fit is with parameters
Pl = 0.5 s-1
Ksp = 10.5 mM2
kp = mM-1s-1

15. a simple theory of precipitation explains the relationship
The steady level of [Ca2+]SR is interpreted as the result of a balance between Ca2+ pump and leak fluxes, Mpump and Ml.
Mpump = - Ml =
(1)
Therefore
(2)
SO42- will lower [Ca2+]SR if the product of [SO42-] and [Ca2+] is greater than a constant of solubility product, Ksp, 37.3 mM2 .
If the permeability of the SR to sulfate is large, [SO42-] in SR will be the same as in the solution, 75 mM, and [Ca2+]SR will be clamped at mM.
in the figure the measured [Ca2+]SR is lower than 0.5 mM and not clamped.
In SO42- the balance equation (1) must be replaced by one that includes a net precipitation flux Mp,
assumed linear on [SO42-]SR and of rate constant kp:
Mpump = - Mleak + Mp =
[Ca2+]SR  Pl + [Ca2+]SR  {[SO42-]SR – Ksp/[Ca2+]SR}  kp (3)
and [Ca2+]SR = Mpump / {Pl + ([SO42-]SR – Ksp/[Ca2+]SR)  kp} (4)
Eqn. 4 can be solved for [Ca2+]SR
(5)
Where [Ca2+]SR(0) represents the value in glutamate, prior to sulfate substitution. This equation applies only when
[Ca2+]SR(0)  [SO42-]SR > Ksp.

16. effects on sparks are unrelated to SR [Ca2+]
10 μm A B C
100 ms D E 2
1.5
glut
SO42-
glut
SO42-
glut G F a b c d e f
012204a

17. sulfate
reference

18. sulfate on reconstituted channels (Copello and Porta)

19. Conclusions so far:
The effects of SO4 on sparks are not mediated by [Ca2+] changes in SR
They are not direct “pharmacological” effects on the RyR
They are probably mediated by cytosolic buffering actions

20. Simulations. Evolution of cytosolic [Ca2+] near an open channel
@ 60 ms
reference
time
space
sulfate, 75mM

21. amplitude
FWHM mm
rise time ms
frequency
s-1
(100 mm)-1
N, events
(cells)
Reference
0.88
0.06
1.53
0.07
5.55
0.41
5.3
2.5
1890
(12)
SO4
75 mM
0.63
0.03
2.02
0.02
7.31
0.32
3.3
1.3
359
(6)
0.83
1.38
3.94
0.15
2.2
408
(3)
PO4
20 mM
0.81
0.01
1.37
3.55
6.3
1335
0.87
1.52
5.35
0.18
522
(2)
Citrate
4 mM
0.85
1.61
5.40
0.13
6.4
683
0.58
5.56
0.12
8.0
1205
(5)
40 mM
0.50
1.48
0.05
4.35
0.28
0.7
174

22. Fluorescence
(spark)
amplit.
rise
time
Ca flux, m 
ampl/rise time
local [Ca2+] = a m

23. A I A model of the Ca release unit Ca2+ ki kr
Let A represent an availability, occupancy of an available state A, which be reduced by an inactivation reaction driven by local Ca with on rate constant ki and recovery rate constant kr.
Local Ca will be proportional to the spark flux, assumed prop. to the ratio amplitude/ rise time.
Therefore
where a is a constant. Because all coefficients are constant, A will evolve as
At a certain time T, A will reach a critical availability A*,
the release unit channels will inactivate and close.
T is the rise time.
Solving:
Where

24. Model fit to reference data
Best fit parameter values
a = 64.3 ms
b = 62.9
What would sulfate do? How would it affect the parameter values?
answer: if its actions are mediated only by Ca buffering it should change b (‘cause it contains alpha) but not a

25. Model fit to data in sulfate
Best fit parameter values
a = 106 ms
b = 177