REGULATION OF CALCIUM RELEASE ACTIVATION AT THE LEVEL OF CALCIUM TRIGGER SIGNALS

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REGULATION OF CALCIUM RELEASE ACTIVATION AT THE
LEVEL OF CALCIUM TRIGGER SIGNALS

• MBI Workshop, March 9, 2004

2
Structure of the myocardial cell
2 µM
courtesy of M Novotova, UMFG SAV

• Architectural features at the scale of 10 nm 2
  µM
• are important for calcium homeostasis

3
Local calcium release
20 µm
Ca (a.u.)
4
The excitation Ca-release coupling unit
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RyR activation by calcium

• Zahradníková et al., J Gen Physiol (2003)

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RyR activation by calcium

• Activation by Ca2 occurs in the H-mode
• At least 4 Ca ions have to bind to the RyR before
  opening

Only H-mode openings (to 2ms)
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Effect of Mg2 on RyR activity
Steady-state

• Xu et al., Circ Res 79 1100-1109, 1996
• Laver et al., J Membr Biol 156 213-229, 1997
• Györke, Györke, Biophys J. 75 2801-2810, 1998
• Kawano, Receptors Channels 5 405-416, 1998

Relative Po
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Competition between Ca2 and Mg2
Ca2 steps 0.1 ? 1 µM Ca2
40 pA
8 pA

• Zahradníková et al., Am J Physiol Cell Physiol
  (2003)

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Competition between Ca2 and Mg2
Ca2 steps 0.1 ? 1 µM Ca2
40 pA
8 pA

• Zahradníková et al., Am J Physiol Cell Physiol
  (2003)

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Competition between Ca2 and Mg2
1.0 mM Mg2
0.5 mM Mg2
Control
100
50
0
Ca2 (mM)
50 pA
0.3
0.0
25 ms
ensemble Po

• Zahradníková et al., Am J Physiol Cell Physiol
  (2003)

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Competition between Ca2 and Mg2
Ca2
Ca2
Ca2
Ca2
H
C
C
C
C
C
O
00
10
20
30
40
1
I
O
C
I
2
5
L
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Competition between Ca2 and Mg2
Ca2
Ca2
Ca2
Ca2
H
C
C
C
C
C
O
00
10
20
30
40
1
I
O
C
I
2
5
L

• The channel can open only if no Mg2 ions are
  bound

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Methods
Scanning line
Patch
-
clamp
Digitizer
Confocal microscope
PC
Synchronizer
20 µm
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Ca2 cross-signaling in the dyadic junction
RDI
5 pA/pF
25 ms
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Ca2 cross-signaling in the dyadic junction
DHPR
RDI
5 pA/pF
25 ms
RyR
Release-dependent inactivation (RDI) as a probe
of junctional Ca
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Calcium release and calcium release-dependent
inactivation of ICa
20 µm
Fpa np / n
prepulse
prepulse tail
test
test tail
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Correlation between Ca2 release and ICa
inactivation
Fpi
R0.838
Fpi
R0.869
0.0
0.5
1.0
0.0
0.5
1.0
Fpa
Fpa

• At the level of individual dyads, inactivation of
  calcium current in the test pulse is directly
  proportional to calcium release activation by the
  prepulse

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MODEL HYPOTHESIS

• Fpi is proportional to the fraction of activated
  release units (Fpa)
• Fpa is dose-dependent on the amount of calcium
  sensed by the RyRs that triggered Ca-release
• Therefore
• Fpi should be dose-dependent on the effective
  integral of ICa

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Effective integral of ICa
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Scheme of the whole-cell experiment
membrane potential
calcium current
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Ca release activation by prepulse and tail current
60
-50
60
1 nA
-50
10 ms
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Ca release activation by tail current only
all openings
Ca release activation

• a1 0.32 ar 1.0

mCa (amol)
Vt (mV)
Fpi
Vt (mV)
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Efficiency depends on previous history

• tail gt prepulse
• reopening gt first opening

• Openings that did not trigger calcium release
  still could
• increase basal Ca2
• saturate Ca buffers

DIRECT TEST using planar lipid bilayers
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Scheme of the BLM experiment
recovery
Ca2 concentration
RyR open probability
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Modulation of RyR activation by basal Ca2
75
50
25
0
Ca2 (mM)
50 pA
Zahradníková et al., Am J Physiol Cell Physiol
(2004)
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INSTEAD OF THE OVERALL CONCLUSIONModel of
calcium release activation

• Single DHPR openings have low probability to
  trigger calcium release
• Triggering probability depends on the recent
  history of DHPR openings (i.e., it increases with
  time)
• The presence of multiple DHPR in the dyad is
  necessary

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Contributors

• ÚMFG SAV, Bratislava, Slovak Republic
• Laboratory of Molecular Biophysics
• Alexandra Zahradníková
• Zuzana Kubalová, Miroslav Dura, Eva Poláková
• Laboratory of Electrophysiology
• Ivan Zahradník
• Jana Pavelková
• Laboratory of Cell Morphology
• Alexandra Zahradníková, jr.
• TTU HSC, Lubbock, TX, USA
• Department of Physiology
• Sándor Györke
• Inna Györke

• Support
• Howard Hughes Medical Institute
• NIH FIRCA (with Sándor Györke)
• Slovak Grant Agency for Science - VEGA

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Timing of prepulse-induced calcium release
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Activation of RyRs

• Po increases with
• stimulus amplitude
• stimulus duration

Open probability
Duration (ms)
Calcium (mM)
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The effect of Mg2 on the RyR activation rateat
different Ca pulse durations
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The effect of Mg2 on RU triggering
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Dependence of RDI on the recovery potential
DHPR recovery ???
1.0
Fpi
0.5
0.0
Vip (mV)

• recovery does not affect Fpi

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Coupling fidelity

• probability that a DHPR opening will activate
  calcium release

• Zhou et al.
• Our method J Physiol 521351, 1999

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BLM / flash photolysis
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Photolysis and complexation of caged Ca2
Ca cage
Ca2
High affinity
Ca cage-Ca2
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Calcium stimuli3 mM NP-EGTA
9 ns flash
Ca2 (mM)
Brief stimulus
20 ms
Sustained stimulus
50 ms