Calcium entry and calcium signaling different routes, different roles

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Calcium entry and calcium signaling different
routes, different roles
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secretion
contraction/motility
metabolism
Ca2
fertilization
apoptosis
differentiation
proliferation
growth
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GPCRs
Gq/11
Ca2
X
Ca2 release (InsP3R)
Ca2 uptake (SERCA)
PLC
DAG InsP3
Ca2
oscillatory behavior of the InsP3R
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GPCRs
PKC
Gq/11
Ca2
Ca2 release (InsP3R)
Ca2 uptake (SERCA)
X
PLC
DAG InsP3
Ca2
oscillations in InsP3 concentrations
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Effect of entry on frequency
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capacitative model
SOC
R
PLC
G
InsP3
Ca
??
Ca
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SOC
R
PLC
G
InsP3
Ca
Ca
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Ca2
R
PLC
PLA2
G
AA
InsP3
Ca
Ca
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Evidence for AA as the second-messenger for
noncapacitative Ca2 entry

• Exogenous administration of low concentrations
  of AA (3-8 mM) induces an entry of Ca2 without
  any detectable depletion of the intracellular
  stores.

• The enzymatic machinery for AA production (e.g.
  a cPLA2) is activated by relevant (low)
  concentrations of the appropriate agonists.

• Inhibition of AA generation prevents the entry
  of Ca2 associated with low concentrations of
  agonists.

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??
Ca2
R
PLC
PLA2
G
AA
InsP3
Ca
Ca
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AA
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AA La3
Naout
AA (8 mM)
m
NMDGout
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Arachidonate-Regulated Calcium channels
ARC channels
HEK-293 cells CHO cells (Yoo et al.,
2000) Parotid acinar cells (mouse human) HeLa
cells COS cells RBL cells DT40 cells
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pharmacology of ARC channels
AA
AA
1 µM 2-APB
1 µM Gd3
5 µM Gd3
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fast inactivation (at 80mV)
ISOC
IARC
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effect of reducing pHo
IARC
ISOC
pH 6.7
pH 6.7
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Currents through ARC and SOC are additive
ISOC
IARC
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effect of arachidonyl CoA
8 µM ACoAinside
8 µM ACoAoutside
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effect of CCh (0.5 mM)
CCh-activated current -
? absolutely dependent on AA generation
? shows no fast inactivation
? NaCa current ratio 40
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effect of CCh conc. on activation of ARC
1 µM CCh
0.2 µM CCh
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LOW agonist concentrations
SOC
ARC
Ca2
X
Ca
Ca
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HIGH agonist concentrations
SOC
Ca2
ARC
Ca2
Ca
Ca
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Mn2 quench rate
TOTAL
isotet
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effect of internal Ca2
SOC
ARC
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HIGH agonist concentrations
SOC
Ca2
ARC
Ca2
Ca
Ca
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agonist conc.
cytosolic Ca2
ARC
Ca2 entry
SOC
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cytosolic Ca2
SOC
ARC
Ca2 entry
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reciprocal regulation of Ca2 entry
- depends on a Ca2-dependent inhibition of ARC
channels
- this is independent of the Ca2 flux through
the channels, and depends on the global
cytosolic Ca2 concentration
- it displays a high sensitivity to Ca2
concentrations above resting values (Kd 150 nM)
- the inhibition by Ca2 is slow ( gt 1 min for
complete inhibition)
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effect of calcineurin inhibitors
control
CspA (1 µM)
ascomycin (5 µM)
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CAIN
(cabin-1)
an endogenous non-competitive inhibitor of
calcineurin
binds calcineurin with high affinity (Ki 450
nM)
expression of the calcineurin binding domain of
CAIN (residues 1881-2173) specifically inhibits
the phosphatase activity of calcineurin
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effect of CAIN
GFP
GFP-c-CAIN
at 400 nM internal Ca2
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GFP-NFAT4-n translocation
0.5 µM CCh for 30 min
10 µM CCh for 15 min
time 0
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NFAT-mediated luciferase expression
control (pCIS-CK luciferase)
NFAT-luciferase
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cytosolic Ca2
SOC
ARC
Ca2 entry
activation of calcineurin
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ARC
SOC
AC
?