Transcript Calcineurin mediates enhanced high-voltage

Calcineurin mediates enhanced high-voltageactivated calcium currents in rat primary cortical
neurons after acute hypoxia
K. Xiang, E.I. Tietz, L.J.Greenfield Jr
Dept. of Internal Medicine, Neurology and
Physiology/Pharmacology,
Univ. of Toledo College of Medicine, Toledo, OH.
Resident symposium April 2010
(From GHAFOORI P et al., ONCOLOGY. Vol. 22 No. 1, 2008.)
Acute oxygen-sensing mechanisms.
Weir EK, López-Barneo J, Buckler KJ, Archer SL.
N Engl J Med. 2005 Nov 10;353(19):2042-55.
• The response of the smoothmuscle cells in the pulmonary
arteries to acute hypoxia
begins within seconds and
involves inhibition of potassium
current, membrane
depolarization, and calcium
entry through L-type calcium
channels; it also involves
calcium release from the
sarcoplasmic reticulum and
calcium repletion through
store-operated channels.
Voltage-Gated Calcium Channels
Table 1. Subunit composition and function of Ca2+ cannel types
Ca 2+
channel
Ca 2+
current
type
CaV1.1
L
CaV1.2
L
CaV1.3
CaV1.4
Primary
localizations
Previous name of
{alpha}1{gamma}
subunits
Specific blocker
Functions
Excitation-contraction coupling
Calcium homeostasis Gene
regulation
Excitation-contraction coupling
Hormone secretion Gene
regulation
Hormone secretion Gene
regulation
{alpha}1S
DHPs
{alpha}1C
DHPs
L
Skeletal muscle
Cardiac muscle
Endocrine cells
Neurons
Endocrine cells
Neurons
{alpha}1D
DHPs
L
Retina
{alpha}1F
CaV2.1
P/Q
Nerve terminals
Dendrites
{alpha}1A
CaV2.2
N
Nerve terminals
Dendrites
{alpha}1B
Tonic neurotransmitter release
Neurotransmittler release Dendritic
{omega}-Agatoxin Ca2+ transients
Neurotransmitter release Dendritic
{omega}-CTx-GVIA Ca2+ transients
CaV2.3
R
CaV3.1
T
CaV3.2
T
CaV3.3
T
Cell bodies Dendrites {alpha}1E
Nerve
Terminals
Cardiac muscle
Skeletal muscle
Neurons
{alpha}1G
Cardiac muscle
Neurons
{alpha}1H
Neurons
{alpha}11
None
Ca2+-dependent action potentials
Neurotransmitter release
None
Repetitive ring
None
Repetitive ring
None
Repetitive ring
Primary cultures of rat cortical neurons
• Primary cortical neuron culture:
13-15 days in vitro culture from
E18 fetal rats.
• Hypoxic exposure with 1% O2 ,
94%N2 and 5%CO2 for 4h;
normoxic exposure (controls)
with 95% air and 5% CO2.
• Recordings were conducted
within 2h of termination of
hypoxia exposure or within ±2
hours after 48h recovery.
Whole-cell Electrophysiology
from Purves et al., 1997
Fig. 1. HVA Ca2+ currents increased immediately after hypoxia
A
Normoxia
Hypoxia
100 pA
100 pA
50 ms
50 ms
B
C
-60
-40
-20
0 mV 20
40
1.4
0
1.2
1.0
G/Gmax
200 ms
pA/pF
-10 +40 mV
-20
0.6
0.4
-80 mV
-30
0.8
Normoxia
Hypoxia
Normoxia
(n=10)
V50 = 0.8  2.0 mV
Slope = 12.3  1.6
Hypoxia
(n=10)
V50 = 1.9  3.7 mV
Slope = 13.2  1.8
0.2
0.0
-80
-60
-40
-20
mV
0
20
40
Inactivation of VGCC
Point mutations in the IQ motif of 77WT affect Ca2+-dependent inactivation.
Nature 399, 159 - 162 (13 May 1999); doi:10.1038/20200
Neuron. 1999 Mar;22(3):549-58.
Fig. 2. Inactivation of HVA Ca2+ currents unchanged after hypoxia
A
Normoxia
Hypoxia
100 pA
100 pA
500 ms
500 ms
B
110
100
Hypoxia
V50 = -37.8  0.4 mV
Slope = 14.1  0.4
90
200 ms
1500 ms
80
+10 mV
I/Imax
70
60
50
40
-80 mV
Normoxia
V50 = -40.3  0.5 mV
Slope = 14.1  0.4
30
20
10
0
-80
-70
-60
-50
mV
-40
-30
-20
-10
0
10
Fig. 3. HVA Ca2+ currents unchanged after 48 h normoxic recovery
B
mV
-60
-40
-20
0
pA/pF
-5
-10
-15
-20
Control
48 hr Recovery
0
20
40
Peak Current Density (pA/pF)
A
25
*
20
15
10
5
0
Normoxia Hypoxia
Normoxia Hypoxia
0 h recovery
48 h recovery
Hypoxia and Calcineurin
•
•
•
Calcineurin (CaN, also termed
protein phosphatase 2B) is a
phosphatase broadly distributed
throughout the body.
Calcineurin promotes hypoxiainducible factor 1alpha expression
by dephosphorylating RACK1 and
blocking RACK1 dimerization. (Liu
et al., 282(51):37064-73. J Biol
Chem. 2007)
Full activation of phosphatase
activity requires the binding of
Ca2+ /calmodulin (CaM) to the
catalytic A subunit of CaN with
concurrent binding of Ca2+ to the
regulatory CaN B subunit.
Calcineurin regulation of neuronal plasticity.
Rachel D. Groth, Robert L. Dunbar and Paul G. Mermelstein
Biochemical and Biophysical Research Communications 311-4, 2003, P1159-1171
• Through direct
dephosphorylation or
disinhibition of PP1, CaN
influences a diverse array of
cellular proteins.
• Green arrows indicate
activating/enhancing
responses; red arrows indicate
inhibitory modulation.
Evaluation of calcineurin in VGCC regulation after hypoxia
• FK-506 (Tacrolimus) and Cyclosporin A (CsA) are structurally
distinct immunosuppressive agents that specifically inhibit
calcineurin activity by binding to separate, endogenously expressed
immunophilins. FK-506 binds to FKBP-12, while CsA binds to
cyclophilin A.
• Okadaic acid is a relatively specific inhibitor of protein
phosphatases 1 and 2A and exhibits little potency toward calcineurin
at drug concentrations of ≤1 μM.
• Rapamycin (Sirolimus) is an immunosuppressant that is similar in
structure to FK-506 and competes for binding to FKBP-12. However,
unlike the FK-506/FKBP-12 complex, the rapamycin/FKBP-12
complex does not bind to and inhibit calcineurin. Thus, rapamycin is
an advantageous agent for separating FK-506’s actions on
immunophilins from its actions on calcineurin.
From Norris et al. (2002) Neuroscience.
Fig. 4. FK506 and CsA reversed the transient HVA Ca2+ current
enhancement after hypoxia
A
D
Normoxia-CsA
Normoxia-FK506
Hypoxia-CsA
100 pA
50 ms
Hypoxia-FK506
E
mV
-60
-40
-20
0
20
40
mV
-60
0
0
-5
-5
pA/pF
pA/pF
B
-10
0
20
40
Normoxia-CsA
Hypoxia-FK506
Hypoxia-CsA
-15
C
F
1.2
1.0
1.0
0.8
0.8
0.8
0.8
0.6
0.6
0.6
0.6
0.4
0.4
0.4
0.4
0.2
0.2
0.2
0.2
0.0
0.0
0.0
1.0
-60
-40
-20
0
mV
20
40
Normoxia-CsA
Hypoxia-CsA
1.2
1.0
0.0
-80
-60
-40
-20
0
mV
20
40
G/Gmax
-80
G/Gmax
1.2
Normoxia-FK506
Hypoxia-FK506
I/Imax
1.2
I/Imax
-20
-10
Normoxia-FK506
-15
-40
Fig. 5. Okadaic acid rapamycin and did not reverse the posthypoxic enhancement of HVA Ca2+ currents
A
D
Normoxia-OKA
Normoxia-RAP
Hypoxia-RAP
Hypoxia-OKA
E
mV
-60
-40
-20
0
20
40
-10
-10
pA/pF
0
-20
F
1.2
-40
-20
0
20
40
Normoxia-RAP
Hypoxia-RAP
-20
Normoxia-OKA
Hypoxia-OKA
C
Normoxia-RAP
Hypoxia-RAP
1.2
1.0
1.0
1.0
0.8
0.8
0.8
0.8
0.6
0.6
0.6
0.6
0.4
0.4
0.4
0.4
0.2
0.2
0.2
0.0
0.0
0.0
-85
-65
-45
-25
mV
-5
15
35
1.2
0.0
-80
-60
-40
-20
mV
0
20
40
G/Gmax
0.2
G/Gmax
1.2
Normoxia-OKA
Hypoxia-OKA
1.0
I/Imax
-60
0
I/Imax
pA/pF
B
100 pA
50 ms
mV
Summary & Conclusions
•
•
•
•
•
•
•
High-voltage activated (HVA) Ca2+ currents were increased ~1.5-fold
immediately after 4 h exposure to 1% O2 but returned to baseline after 48 h
normoxic recovery.
The half-maximal potentials of activation and steady-state inactivation were
unchanged.
The calcineurin inhibitor FK506 (5 mM in the recording pipette) reversed
the post-hypoxic increase in VGCC current.
Exposure to a structurally different calcineurin inhibitor, cyclosporine A (20
mM), during hypoxia blocked the increase in VGCC current.
Rapamycin, a FK506 analog that does not block calcineurin activity, failed
to reverse the post-hypoxic increase in VGCC current.
Okadaic acid, an inhibitor of PP1 and PP2A, failed to prevent the posthypoxic increase in VGCC current, suggesting that VGCC regulation is
calcineurin-specifc.
In summary, hypoxia transiently upregulated HVA VGCC currents in
primary cortical neurons via a calcium dependent process involving
calcineurin, suggesting a positive feedback loop to amplify neuronal calcium
signaling after hypoxia.