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
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(From GHAFOORI P et al. , ONCOLOGY. Vol. 22 No. 1, 2008.)
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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.
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
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500 ms
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HypoxiaV50 = -37.8 0.4 mVSlope = 14.1 0.4
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ax
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+10 mV1500 ms
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Fig. 3. HVA Ca2+ currents unchanged after 48 h normoxic recovery
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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
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Fig. 5. Okadaic acid rapamycin and did not reverse the post-hypoxic enhancement of HVA Ca2+ currents
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• 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.