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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Atypical L-type channels are down-regulated in hypoxia
L Callinan1, T V McCarthy, Y Maulet
1Department of Biochemistry, University College Cork, Cork, Ireland. lauracallinan@yahoo.com
Abstract:
One type of cellular response to hypoxia is an increase in cytosolic Ca2+. VDCCs (voltage-dependent calcium channels) open upon membrane depolarization allowing inward current of Ca2+ ions. Two of the so-called L-type VDCC alpha1 subunits, Ca(v)1.2 and Ca(v)1.3, are found in the brain. We sought to investigate the effect of chronic hypoxia or treatment with a hypoxia-mimicking agent DFX (desferrioxamine mesylate) on expression of L-type VDCC in the SH-SY5Y neuroblastoma cell line. Western blotting identified two atypical forms of the L-type channel with apparent molecular masses of approx. 100 and 150 kDa, compared with typical forms of approx. 200 kDa. Immunofluorescence microscopy shows the approx. 100 kDa protein located within the cell and on the cell surface, while the approx. 150 kDa protein is intracellular with punctate staining. Further analysis revealed that this approx. 150 kDa protein co-localizes with nuclear proteins but not with markers for other intracellular compartments. In addition, these proteins are both down-regulated in DFX-treated and hypoxic cells, suggesting that the mechanism of down-regulation is along the HIF (hypoxia-inducible factor) pathway. This atypical localization of the 150 kDa protein suggests that it might play a role in nuclear calcium signalling in health and disease.
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