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

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

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

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)

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

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

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

SEER and other studies of last three years

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

REFERENCE SULFATE WASHOUT A B C b c a

REFERENCE PHOSPHATE WASHOUT A B C b a c

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

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

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 = 0.009 mM-1s-1

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 0.497 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.

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

sulfate reference

sulfate on reconstituted channels (Copello and Porta)

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

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

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

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

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

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

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