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 potentialsNerve 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 Electrophysiology

Fig. 1. HVA Ca2+ currents increased immediately after hypoxia

100 pA50 ms

A

B C-60 -40 -20 0 20 40

-30

-20

-10

0

NormoxiaHypoxia

pA/p

F

mV

-80 mV

+40 mV200 ms

100 pA50 ms

Normoxia Hypoxia

-80 -60 -40 -20 0 20 400.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 Normoxia(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

102030405060708090

100110

NormoxiaV50 = -40.3 0.5 mVSlope = 14.1 0.4

mV

HypoxiaV50 = -37.8 0.4 mVSlope = 14.1 0.4

I/Im

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 RecoveryControl

pA/p

F

Normoxia Hypoxia Normoxia Hypoxia0

5

10

15

20

25

0 h recovery 48 h recovery

*

Peak

Cur

rent

Den

sity

(pA

/pF)

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-FK506Hypoxia-FK506

mV

pA/p

F

-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

max

Normoxia-FK506

Hypoxia-FK506

-80 -60 -40 -20 0 20 400.0

0.2

0.4

0.6

0.8

1.0

1.2Hypoxia-CsA

0.0

0.2

0.4

0.6

0.8

1.0

1.2Normoxia-CsA

mV

I/Im

ax

G/G

max

-60 -40 -20 0 20 40

-15

-10

-5

0

Hypoxia-CsA

mV

Normoxia-CsA

pA/p

F

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/p

F

-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

max

100 pA50 ms

Normoxia-RAP

Hypoxia-RAP

-60 -40 -20 0 20 40

-20

-10

0

mV

Normoxia-OKAHypoxia-OKA

pA/p

F

-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

max

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