Cells take up transferrin-bound iron or NTBI (non-transferrin-bound iron). After treatment with NGF (nerve growth factor), PC12 cells exhibited a neuronal phenotype and an increase in the NTBI uptake (55Fe2+ or 55Fe3+). We loaded the cells with the dye calcein, whose fluorescence increases in the presence of Ca2+ but is quenched with Fe2+ or Fe3+. When examined using calcein fluorescence or radioactive iron, DAG (diacylglycerol)-stimulated NTBI entry was more in NGF-treated PC12 cells compared with untreated cells. All experiments were performed at 1.5 mM extracellular Ca2+. Nramp2 (natural-resistance-associated macrophage protein 2) mRNA expression did not change after the NGF treatment. Expression of the bivalent cation entry protein TRPC6 (transient receptor potential canonical 6) was detected only in the NGF-treated cells. To verify that increased NTBI uptake depended on TRPC6, we examined whether transfecting HEK-293 (human embryonic kidney 293) cells with TRPC6 also increased the NTBI (55Fe) uptake. We also cotransfected HEK-293 cells with two plasmids, one expressing TRPC6 and the other expressing the fluorescent protein DsRED2 to identify the transfected cells. Challenging the calcein-loaded HEK-293 cells (which intrinsically express the α1-adrenergic receptors) with phenylephrine or a cell-permeant DAG increased the fluorescence signal more rapidly in transfected cells compared with untransfected cells. However, when iron (Fe2+ and Fe3+) was added before adding phenylephrine or DAG, the fluorescence intensity decreased more rapidly in transfected cells compared with untransfected cells, thereby indicating a greater stimulation of the NTBI uptake in cells expressing TRPC6. We postulate that the increase in the NTBI entry into neuronal PC12 cells is through TRPC6, a pathway that is unique since it is receptor-stimulated. Since neuronal cells express TRPC6, this pathway may have a role in neurotoxicity.
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March 2004
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Research Article|
March 15 2004
Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells
James MWANJEWE;
James MWANJEWE
Department of Medicine, HSC 4N41, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5
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Ashok K. GROVER
Ashok K. GROVER
1
Department of Medicine, HSC 4N41, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5
1To whom correspondence should be addressed (e-mail groverak@mcmaster.ca).
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Publisher: Portland Press Ltd
Received:
August 05 2003
Revision Received:
October 22 2003
Accepted:
November 26 2003
Accepted Manuscript online:
November 26 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 378 (3): 975–982.
Article history
Received:
August 05 2003
Revision Received:
October 22 2003
Accepted:
November 26 2003
Accepted Manuscript online:
November 26 2003
Citation
James MWANJEWE, Ashok K. GROVER; Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells. Biochem J 15 March 2004; 378 (3): 975–982. doi: https://doi.org/10.1042/bj20031187
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