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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.