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Calcineurin mediates enhanced high-voltage-activated 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 smooth-
muscle 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
Ca2+
channel
Ca2+
current type
Primary localizations
Previous name of {alpha}1{gamma}
subunitsSpecific blocker Functions
CaV1.1 L Skeletal muscle {alpha}1S DHPs
Excitation-contraction coupling Calcium homeostasis Gene regulation
CaV1.2 L
Cardiac muscle Endocrine cells Neurons {alpha}1C DHPs
Excitation-contraction coupling Hormone secretion Gene regulation
CaV1.3 LEndocrine cells Neurons {alpha}1D DHPs
Hormone secretion Gene regulation
CaV1.4 L Retina {alpha}1F Tonic neurotransmitter release
CaV2.1 P/QNerve terminals Dendrites {alpha}1A {omega}-Agatoxin
Neurotransmittler release Dendritic
Ca2+ transients
CaV2.2 NNerve terminals Dendrites {alpha}1B {omega}-CTx-GVIA
Neurotransmitter release Dendritic
Ca2+ transients
CaV2.3 R Cell bodies Dendrites {alpha}1E None Ca2+-dependent action potentials
Nerve Neurotransmitter releaseTerminals
CaV3.1 T
Cardiac muscle Skeletal muscle Neurons {alpha}1G None Repetitive ring
CaV3.2 TCardiac muscle Neurons {alpha}1H None Repetitive ring
CaV3.3 T Neurons {alpha}11 None Repetitive ring
Table 1. Subunit composition and function of Ca2+ cannel types
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.
from Purves et al., 1997
Whole-cell ElectrophysiologyWhole-cell Electrophysiology
Fig. 1. HVA Ca2+ currents increased immediately after hypoxia
100 pA
50 ms
A
B C-60 -40 -20 0 20 40
-30
-20
-10
0
NormoxiaHypoxia
pA
/pF
mV
-80 mV
+40 mV200 ms
100 pA
50 ms
Normoxia Hypoxia
-80 -60 -40 -20 0 20 400.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4Normoxia(n=10)V50 = 0.8 2.0 mVSlope = 12.3 1.6
Hypoxia(n=10)V50 = 1.9 3.7 mVSlope = 13.2 1.8
mV
G/G
max
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
100 pA
500 ms
100 pA
500 ms
-80 -70 -60 -50 -40 -30 -20 -10 0 100
10
20
30
40
50
60
70
80
90
100
110
NormoxiaV50 = -40.3 0.5 mVSlope = 14.1 0.4
mV
HypoxiaV50 = -37.8 0.4 mVSlope = 14.1 0.4
I/I m
ax
-80 mV
+10 mV1500 ms
200 ms
A
B
Normoxia Hypoxia
Fig. 3. HVA Ca2+ currents unchanged after 48 h normoxic recovery
-60 -40 -20 0 20 40
-20
-15
-10
-5
0
mV
48 hr Recovery
Control
pA
/pF
Normoxia Hypoxia Normoxia Hypoxia0
5
10
15
20
25
0 h recovery 48 h recovery
*
Pea
k C
urr
ent
Den
sity
(p
A/p
F)
A B
Hypoxia and Calcineurin
• Calcineurin (CaN, also termed protein phosphatase 2B) is a phosphatase broadly distributed throughout the body.
• Calcineurin promotes hypoxia-inducible 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
B E
C F
-60 -40 -20 0 20 40
-15
-10
-5
0
Normoxia-FK506
Hypoxia-FK506
mV
pA
/pF
-80 -60 -40 -20 0 20 400.0
0.2
0.4
0.6
0.8
1.0
1.2 Normoxia-FK506Hypoxia-FK506
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mV
I/Im
ax
G/G
ma
x
Normoxia-FK506
Hypoxia-FK506
-80 -60 -40 -20 0 20 400.0
0.2
0.4
0.6
0.8
1.0
1.2
Hypoxia-CsA
0.0
0.2
0.4
0.6
0.8
1.0
1.2Normoxia-CsA
mV
I/Im
ax
G/G
ma
x
-60 -40 -20 0 20 40
-15
-10
-5
0
Hypoxia-CsA
mV
Normoxia-CsA
pA
/pF
100 pA
50 ms
Normoxia-CsA
Hypoxia-CsA
Fig. 5. Okadaic acid rapamycin and did not reverse the post-hypoxic enhancement of HVA Ca2+ currents
A D
B E
C F
-60 -40 -20 0 20 40
-20
-10
0
mV
Hypoxia-RAPNormoxia-RAP
pA
/pF
-80 -60 -40 -20 0 20 400.0
0.2
0.4
0.6
0.8
1.0
1.2 Normoxia-RAPHypoxia-RAP
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mV
I/Im
ax
G/G
ma
x
100 pA
50 ms
Normoxia-RAP
Hypoxia-RAP
-60 -40 -20 0 20 40
-20
-10
0
mV
Normoxia-OKAHypoxia-OKA
pA
/pF
-85 -65 -45 -25 -5 15 350.0
0.2
0.4
0.6
0.8
1.0
1.2 Normoxia-OKAHypoxia-OKA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mV
I/Im
ax
G/G
ma
x
Normoxia-OKA
Hypoxia-OKA
• 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 post-hypoxic 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.
Summary & Conclusions
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