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Biochem J. Mar 15, 2004; 378(Pt 3): 975–982.
PMCID: PMC1224001

Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells.

Abstract

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 a1-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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Missiaen L, Wuytack F, Raeymaekers L, De Smedt H, Droogmans G, Declerck I, Casteels R. Ca2+ extrusion across plasma membrane and Ca2+ uptake by intracellular stores. Pharmacol Ther. 1991;50(2):191–232. [PubMed]
  • Huber A, Sander P, Gobert A, Bähner M, Hermann R, Paulsen R. The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD. EMBO J. 1996 Dec 16;15(24):7036–7045. [PMC free article] [PubMed]
  • Hurst RS, Zhu X, Boulay G, Birnbaumer L, Stefani E. Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells. FEBS Lett. 1998 Feb 6;422(3):333–338. [PubMed]
  • McKay RR, Szymeczek-Seay CL, Lievremont JP, Bird GS, Zitt C, Jüngling E, Lückhoff A, Putney JW., Jr Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3. Biochem J. 2000 Nov 1;351(Pt 3):735–746. [PMC free article] [PubMed]
  • Montell Craig, Birnbaumer Lutz, Flockerzi Veit, Bindels René J, Bruford Elspeth A, Caterina Michael J, Clapham David E, Harteneck Christian, Heller Stefan, Julius David, et al. A unified nomenclature for the superfamily of TRP cation channels. Mol Cell. 2002 Feb;9(2):229–231. [PubMed]
  • Hassock Sheila R, Zhu Michael X, Trost Claudia, Flockerzi Veit, Authi Kalwant S. Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel. Blood. 2002 Oct 15;100(8):2801–2811. [PubMed]
  • Wu Xiaoyan, Babnigg György, Zagranichnaya Tatiana, Villereal Mitchel L. The role of endogenous human Trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells. J Biol Chem. 2002 Apr 19;277(16):13597–13608. [PubMed]
  • Zhang L, Saffen D. Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization. J Biol Chem. 2001 Apr 20;276(16):13331–13339. [PubMed]
  • Boulay G, Zhu X, Peyton M, Jiang M, Hurst R, Stefani E, Birnbaumer L. Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J Biol Chem. 1997 Nov 21;272(47):29672–29680. [PubMed]
  • Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature. 1999 Jan 21;397(6716):259–263. [PubMed]
  • Sylvester JB, Mwanjewe J, Grover AK. Transient receptor potential protein mRNA expression in rat substantia nigra. Neurosci Lett. 2001 Mar 9;300(2):83–86. [PubMed]
  • Lintschinger B, Balzer-Geldsetzer M, Baskaran T, Graier WF, Romanin C, Zhu MX, Groschner K. Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels. J Biol Chem. 2000 Sep 8;275(36):27799–27805. [PubMed]
  • Foder B, Scharff O, Thastrup O. Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin. Cell Calcium. 1989 Oct;10(7):477–490. [PubMed]
  • Zhu X, Jiang M, Birnbaumer L. Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry. J Biol Chem. 1998 Jan 2;273(1):133–142. [PubMed]
  • Kaplan Jerry. Mechanisms of cellular iron acquisition: another iron in the fire. Cell. 2002 Nov 27;111(5):603–606. [PubMed]
  • Richardson DR, Ponka P. The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta. 1997 Mar 14;1331(1):1–40. [PubMed]
  • Atkinson PG, Blackwell JM, Barton CH. Nramp1 locus encodes a 65 kDa interferon-gamma-inducible protein in murine macrophages. Biochem J. 1997 Aug 1;325(Pt 3):779–786. [PMC free article] [PubMed]
  • Canonne-Hergaux F, Gruenheid S, Ponka P, Gros P. Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood. 1999 Jun 15;93(12):4406–4417. [PubMed]
  • Garrick LM, Dolan KG, Romano MA, Garrick MD. Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells. J Cell Physiol. 1999 Mar;178(3):349–358. [PubMed]
  • Wardrop SL, Richardson DR. The effect of intracellular iron concentration and nitrogen monoxide on Nramp2 expression and non-transferrin-bound iron uptake. Eur J Biochem. 1999 Jul;263(1):41–49. [PubMed]
  • Zoller H, Pietrangelo A, Vogel W, Weiss G. Duodenal metal-transporter (DMT-1, NRAMP-2) expression in patients with hereditary haemochromatosis. Lancet. 1999 Jun 19;353(9170):2120–2123. [PubMed]
  • McKie AT, Latunde-Dada GO, Miret S, McGregor JA, Anderson GJ, Vulpe CD, Wrigglesworth JM, Simpson RJ. Molecular evidence for the role of a ferric reductase in iron transport. Biochem Soc Trans. 2002 Aug;30(4):722–724. [PubMed]
  • Conrad ME, Umbreit JN, Moore EG, Hainsworth LN, Porubcin M, Simovich MJ, Nakada MT, Dolan K, Garrick MD. Separate pathways for cellular uptake of ferric and ferrous iron. Am J Physiol Gastrointest Liver Physiol. 2000 Oct;279(4):G767–G774. [PubMed]
  • Ahnert-Hilger G, Gratzl M. Further characterization of dopamine release by permeabilized PC12 cells. J Neurochem. 1987 Sep;49(3):764–770. [PubMed]
  • Bouron A, Becker C, Porzig H. Functional expression of voltage-gated Na+ and Ca2+ channels during neuronal differentiation of PC12 cells with nerve growth factor or forskolin. Naunyn Schmiedebergs Arch Pharmacol. 1999 May;359(5):370–377. [PubMed]
  • Mwanjewe J, Hui BK, Coughlin MD, Grover AK. Treatment of PC12 cells with nerve growth factor increases iron uptake. Biochem J. 2001 Aug 1;357(Pt 3):881–886. [PMC free article] [PubMed]
  • Phung YT, Bekker JM, Hallmark OG, Black SM. Both neuronal NO synthase and nitric oxide are required for PC12 cell differentiation: a cGMP independent pathway. Brain Res Mol Brain Res. 1999 Feb 5;64(2):165–178. [PubMed]
  • Mwanjewe J, Martinez R, Agrawal P, Samson SE, Coughlin MD, Brassard P, Grover AK. On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells. J Biol Chem. 2000 Oct 27;275(43):33512–33515. [PubMed]
  • Epsztejn S, Kakhlon O, Glickstein H, Breuer W, Cabantchik I. Fluorescence analysis of the labile iron pool of mammalian cells. Anal Biochem. 1997 May 15;248(1):31–40. [PubMed]
  • Thomas F, Serratrice G, Béguin C, Aman ES, Pierre JL, Fontecave M, Laulhère JP. Calcein as a fluorescent probe for ferric iron. Application to iron nutrition in plant cells. J Biol Chem. 1999 May 7;274(19):13375–13383. [PubMed]
  • Gruenheid S, Canonne-Hergaux F, Gauthier S, Hackam DJ, Grinstein S, Gros P. The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes. J Exp Med. 1999 Mar 1;189(5):831–841. [PMC free article] [PubMed]
  • Belmeguenai Amor, Leprince Jérôme, Tonon Marie-Christine, Vaudry Hubert, Louiset Estelle. Neurotensin modulates the amplitude and frequency of voltage-activated Ca2+ currents in frog pituitary melanotrophs: implication of the inositol triphosphate/protein kinase C pathway. Eur J Neurosci. 2002 Nov;16(10):1907–1916. [PubMed]
  • Delumeau Jean C, Tencé Martine, Marin Philippe, Cordier Jocelyne, Glowinski Jacques, Prémont Joël. Synergistic Regulation of Cytosolic Ca2+ Concentration by Adenosine and alpha1-Adrenergic Agonists in Mouse Striatal Astrocytes. Eur J Neurosci. 1991 Jun;3(6):539–550. [PubMed]
  • Marsh SJ, Trouslard J, Leaney JL, Brown DA. Synergistic regulation of a neuronal chloride current by intracellular calcium and muscarinic receptor activation: a role for protein kinase C. Neuron. 1995 Sep;15(3):729–737. [PubMed]
  • Sigel E, Baur R. Activation of protein kinase C differentially modulates neuronal Na+, Ca2+, and gamma-aminobutyrate type A channels. Proc Natl Acad Sci U S A. 1988 Aug;85(16):6192–6196. [PMC free article] [PubMed]
  • Stemkowski Patrick L, Tse Frederick W, Peuckmann Vera, Ford Christopher P, Colmers William F, Smith Peter A. ATP-inhibition of M current in frog sympathetic neurons involves phospholipase C but not Ins P(3), Ca(2+), PKC, or Ras. J Neurophysiol. 2002 Jul;88(1):277–288. [PubMed]
  • Chen R, Greene EL, Collinsworth G, Grewal JS, Houghton O, Zeng H, Garnovskaya M, Paul RV, Raymond JR. Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP. Am J Physiol. 1999 May;276(5 Pt 2):F777–F785. [PubMed]
  • Kovala AT, Harvey KA, McGlynn P, Boguslawski G, Garcia JG, English D. High-efficiency transient transfection of endothelial cells for functional analysis. FASEB J. 2000 Dec;14(15):2486–2494. [PubMed]
  • Inoue R, Okada T, Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y, Mori Y. The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel. Circ Res. 2001 Feb 16;88(3):325–332. [PubMed]
  • Goetz David H, Holmes Margaret A, Borregaard Niels, Bluhm Martin E, Raymond Kenneth N, Strong Roland K. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell. 2002 Nov;10(5):1033–1043. [PubMed]
  • Roth Jerome A, Horbinski Craig, Higgins Dennis, Lein Pamela, Garrick Michael D. Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation. Neurotoxicology. 2002 Jul;23(2):147–157. [PubMed]
  • Yang Jun, Goetz David, Li Jau Yi, Wang Wenge, Mori Kiyoshi, Setlik Daria, Du Tonggong, Erdjument-Bromage Hediye, Tempst Paul, Strong Roland, et al. An iron delivery pathway mediated by a lipocalin. Mol Cell. 2002 Nov;10(5):1045–1056. [PubMed]
  • Faucheux BA, Hirsch EC, Villares J, Selimi F, Mouatt-Prigent A, Javoy-Agid F, Hauw JJ, Agid Y. Distribution of 125I-ferrotransferrin binding sites in the mesencephalon of control subjects and patients with Parkinson's disease. J Neurochem. 1993 Jun;60(6):2338–2341. [PubMed]
  • Hirsch EC, Faucheux BA. Iron metabolism and Parkinson's disease. Mov Disord. 1998;13 (Suppl 1):39–45. [PubMed]

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