1. 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

2. (From GHAFOORI P et al., ONCOLOGY. Vol. 22 No. 1, 2008.)

3. 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.

4. Voltage-Gated Calcium Channels

6. Primary cultures of rat cortical neurons Primary cortical neuron culture: 13-15 days in vitro culture from E18 fetal rats. Hypoxic exposure with 1% O 2, 94%N 2 and 5%CO 2 for 4h; normoxic exposure (controls) with 95% air and 5% CO 2. Recordings were conducted within 2h of termination of hypoxia exposure or within ±2 hours after 48h recovery.

7. from Purves et al., 1997 Whole-cell Electrophysiology

8. Fig. 1. HVA Ca 2+ currents increased immediately after hypoxia

9. 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.

10. Fig. 2. Inactivation of HVA Ca 2+ currents unchanged after hypoxia

11. Fig. 3. HVA Ca 2+ currents unchanged after 48 h normoxic recovery

12. 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 Ca 2+ /calmodulin (CaM) to the catalytic A subunit of CaN with concurrent binding of Ca 2+ to the regulatory CaN B subunit.

13. 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.

14. 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.

15. Fig. 4. FK506 and CsA reversed the transient HVA Ca 2+ current enhancement after hypoxia

16. Fig. 5. Okadaic acid rapamycin and did not reverse the post- hypoxic enhancement of HVA Ca 2+ currents

17. High-voltage activated (HVA) Ca 2+ 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