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. 2001 Sep 15;358(Pt 3):717–726. doi: 10.1042/0264-6021:3580717

A diacylglycerol-activated Ca2+ channel in PC12 cells (an adrenal chromaffin cell line) correlates with expression of the TRP-6 (transient receptor potential) protein.

Y Tesfai 1, H M Brereton 1, G J Barritt 1
PMCID: PMC1222105  PMID: 11535132

Abstract

The structures, and mechanisms of activation, of plasma membrane intracellular-messenger-activated, non-selective cation channels in animal cells are not well understood. The PC12 adrenal chromaffin cell line is a well-characterized example of a nerve cell. In PC12 cells, 1-oleolyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, initiated the inflow of Ca(2+), Mn(2+) and Sr(2+). Acetylcholine and thapsigargin initiated the inflow of Ca(2+) and Mn(2+), but not of Sr(2+). The activation of bivalent cation inflow by OAG: (i) was mimicked by another membrane-permeant diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, but not by the membrane-impermeant analogue 1-stearoyl-2-arachidonyl-sn-glycerol; (ii) was not blocked by staurosporin or chelerythrine, inhibitors of protein kinase C; (iii) was enhanced by RHC80267 and R50922, inhibitors of diacylglycerol lipase and diacylglycerol kinase respectively; and (iv) was inhibited by extracellular Ca(2+). When OAG was added after acetylcholine, the effect of OAG on Ca(2+) inflow was over-and-above that induced by acetylcholine. 2-Aminoethyl diphenylborate (2-APB) inhibited Ca(2+) inflow initiated by either acetylcholine or thapsigargin, but not that initiated by OAG. Flufenamic acid inhibited OAG-initiated, but not acetylcholine-initiated, Ca(2+) and Mn(2+) inflow. OAG-initiated Ca(2+) inflow was less sensitive to inhibition by SK&F96365 than acetylcholine-initiated Ca(2+) inflow. In polyadenylated RNA prepared from PC12 cells, mRNA encoding TRP (transient receptor potential) proteins 1-6 was detected by reverse transcriptase (RT)-PCR, and in lysates of PC12 cells the endogenous TRP-6 protein was detected by Western blot analysis. It is concluded that PC12 cells express a diacylglycerol-activated, non-selective cation channel. Expression of this channel function correlates with expression of the TRP-3 and TRP-6 proteins, which have been shown previously to be activated by diacylglycerol when expressed heterologously in animal cells [Hofmann, Obukhov, Schaefer, Harteneck, Gudermann, and Schultz (1999) Nature (London) 397, 259-263].

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Selected References

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  1. Altin J. G., Bradshaw R. A. Production of 1,2-diacylglycerol in PC12 cells by nerve growth factor and basic fibroblast growth factor. J Neurochem. 1990 May;54(5):1666–1676. doi: 10.1111/j.1471-4159.1990.tb01220.x. [DOI] [PubMed] [Google Scholar]
  2. Auld A., Chen J., Brereton H. M., Wang Y. J., Gregory R. B., Barritt G. J. Store-operated Ca(2+) inflow in Reuber hepatoma cells is inhibited by voltage-operated Ca(2+) channel antagonists and, in contrast to freshly isolated hepatocytes, does not require a pertussis toxin-sensitive trimeric GTP-binding protein. Biochim Biophys Acta. 2000 Jun 2;1497(1):11–26. doi: 10.1016/s0167-4889(00)00045-8. [DOI] [PubMed] [Google Scholar]
  3. Barritt G. J. Receptor-activated Ca2+ inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2+ signalling requirements. Biochem J. 1999 Jan 15;337(Pt 2):153–169. [PMC free article] [PubMed] [Google Scholar]
  4. Bennett D. L., Bootman M. D., Berridge M. J., Cheek T. R. Ca2+ entry into PC12 cells initiated by ryanodine receptors or inositol 1,4,5-trisphosphate receptors. Biochem J. 1998 Jan 15;329(Pt 2):349–357. doi: 10.1042/bj3290349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berridge M. J. Neuronal calcium signaling. Neuron. 1998 Jul;21(1):13–26. doi: 10.1016/s0896-6273(00)80510-3. [DOI] [PubMed] [Google Scholar]
  6. 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. doi: 10.1074/jbc.272.47.29672. [DOI] [PubMed] [Google Scholar]
  7. Braun F. J., Broad L. M., Armstrong D. L., Putney J. W., Jr Stable activation of single Ca2+ release-activated Ca2+ channels in divalent cation-free solutions. J Biol Chem. 2001 Jan 12;276(2):1063–1070. doi: 10.1074/jbc.M008348200. [DOI] [PubMed] [Google Scholar]
  8. Brereton H. M., Harland M. L., Froscio M., Petronijevic T., Barritt G. J. Novel variants of voltage-operated calcium channel alpha 1-subunit transcripts in a rat liver-derived cell line: deletion in the IVS4 voltage sensing region. Cell Calcium. 1997 Jul;22(1):39–52. doi: 10.1016/s0143-4160(97)90088-9. [DOI] [PubMed] [Google Scholar]
  9. Buess M., Engler O., Hirsch H. H., Moroni C. Search for oncogenic regulators in an autocrine tumor model using differential display PCR: identification of novel candidate genes including the calcium channel mtrp6. Oncogene. 1999 Feb 18;18(7):1487–1494. doi: 10.1038/sj.onc.1202445. [DOI] [PubMed] [Google Scholar]
  10. Clementi E., Scheer H., Zacchetti D., Fasolato C., Pozzan T., Meldolesi J. Receptor-activated Ca2+ influx. Two independently regulated mechanisms of influx stimulation coexist in neurosecretory PC12 cells. J Biol Chem. 1992 Feb 5;267(4):2164–2172. [PubMed] [Google Scholar]
  11. Di Virgilio F., Fasolato C., Steinberg T. H. Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells. Biochem J. 1988 Dec 15;256(3):959–963. doi: 10.1042/bj2560959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Egea J., Espinet C., Comella J. X. Calcium influx activates extracellular-regulated kinase/mitogen-activated protein kinase pathway through a calmodulin-sensitive mechanism in PC12 cells. J Biol Chem. 1999 Jan 1;274(1):75–85. doi: 10.1074/jbc.274.1.75. [DOI] [PubMed] [Google Scholar]
  13. Estacion M., Sinkins W. G., Schilling W. P. Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms. J Physiol. 2001 Jan 1;530(Pt 1):1–19. doi: 10.1111/j.1469-7793.2001.0001m.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garcia R. L., Schilling W. P. Differential expression of mammalian TRP homologues across tissues and cell lines. Biochem Biophys Res Commun. 1997 Oct 9;239(1):279–283. doi: 10.1006/bbrc.1997.7458. [DOI] [PubMed] [Google Scholar]
  15. García M. C., López M. G., García A. G., Sánchez Crespo M. Muscarinic acetylcholine receptor enhances phosphatidylcholine hydrolysis via phospholipase D in bovine chromaffin cells in culture. J Neurochem. 1992 Dec;59(6):2244–2250. doi: 10.1111/j.1471-4159.1992.tb10117.x. [DOI] [PubMed] [Google Scholar]
  16. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gregory R. B., Rychkov G., Barritt G. J. Evidence that 2-aminoethyl diphenylborate is a novel inhibitor of store-operated Ca2+ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors. Biochem J. 2001 Mar 1;354(Pt 2):285–290. doi: 10.1042/0264-6021:3540285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gögelein H., Dahlem D., Englert H. C., Lang H. J. Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas. FEBS Lett. 1990 Jul 30;268(1):79–82. doi: 10.1016/0014-5793(90)80977-q. [DOI] [PubMed] [Google Scholar]
  19. Harteneck C., Plant T. D., Schultz G. From worm to man: three subfamilies of TRP channels. Trends Neurosci. 2000 Apr;23(4):159–166. doi: 10.1016/s0166-2236(99)01532-5. [DOI] [PubMed] [Google Scholar]
  20. Helliwell R. M., Large W. A. Alpha 1-adrenoceptor activation of a non-selective cation current in rabbit portal vein by 1,2-diacyl-sn-glycerol. J Physiol. 1997 Mar 1;499(Pt 2):417–428. doi: 10.1113/jphysiol.1997.sp021938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Herbert J. M., Augereau J. M., Gleye J., Maffrand J. P. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990 Nov 15;172(3):993–999. doi: 10.1016/0006-291x(90)91544-3. [DOI] [PubMed] [Google Scholar]
  22. Hofmann T., Obukhov A. G., 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. doi: 10.1038/16711. [DOI] [PubMed] [Google Scholar]
  23. Hofmann T., Schaefer M., Schultz G., Gudermann T. Transient receptor potential channels as molecular substrates of receptor-mediated cation entry. J Mol Med (Berl) 2000;78(1):14–25. doi: 10.1007/s001099900070. [DOI] [PubMed] [Google Scholar]
  24. Horwitz J. Carbachol and bradykinin increase the production of diacylglycerol from sources other than inositol-containing phospholipids in PC12 cells. J Neurochem. 1990 Mar;54(3):983–991. doi: 10.1111/j.1471-4159.1990.tb02347.x. [DOI] [PubMed] [Google Scholar]
  25. Kamouchi M., Philipp S., Flockerzi V., Wissenbach U., Mamin A., Raeymaekers L., Eggermont J., Droogmans G., Nilius B. Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells. J Physiol. 1999 Jul 15;518(Pt 2):345–358. doi: 10.1111/j.1469-7793.1999.0345p.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li H. S., Xu X. Z., Montell C. Activation of a TRPC3-dependent cation current through the neurotrophin BDNF. Neuron. 1999 Sep;24(1):261–273. doi: 10.1016/s0896-6273(00)80838-7. [DOI] [PubMed] [Google Scholar]
  27. Lintschinger B., Balzer-Geldsetzer M., Baskaran T., Graier W. F., Romanin C., Zhu M. X., 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. doi: 10.1074/jbc.M002705200. [DOI] [PubMed] [Google Scholar]
  28. Ma H. T., Patterson R. L., van Rossum D. B., Birnbaumer L., Mikoshiba K., Gill D. L. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science. 2000 Mar 3;287(5458):1647–1651. doi: 10.1126/science.287.5458.1647. [DOI] [PubMed] [Google Scholar]
  29. Magoski N. S., Knox R. J., Kaczmarek L. K. Activation of a Ca2+-permeable cation channel produces a prolonged attenuation of intracellular Ca2+ release in Aplysia bag cell neurones. J Physiol. 2000 Jan 15;522(Pt 2):271–283. doi: 10.1111/j.1469-7793.2000.t01-2-00271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Merritt J. E., Armstrong W. P., Benham C. D., Hallam T. J., Jacob R., Jaxa-Chamiec A., Leigh B. K., McCarthy S. A., Moores K. E., Rink T. J. SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J. 1990 Oct 15;271(2):515–522. doi: 10.1042/bj2710515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mizuno N., Kitayama S., Saishin Y., Shimada S., Morita K., Mitsuhata C., Kurihara H., Dohi T. Molecular cloning and characterization of rat trp homologues from brain. Brain Res Mol Brain Res. 1999 Jan 22;64(1):41–51. doi: 10.1016/s0169-328x(98)00296-4. [DOI] [PubMed] [Google Scholar]
  32. Mizuno N., Kitayama S., Saishin Y., Shimada S., Morita K., Mitsuhata C., Kurihara H., Dohi T. Molecular cloning and characterization of rat trp homologues from brain. Brain Res Mol Brain Res. 1999 Jan 22;64(1):41–51. doi: 10.1016/s0169-328x(98)00296-4. [DOI] [PubMed] [Google Scholar]
  33. Okada T., Inoue R., Yamazaki K., Maeda A., Kurosaki T., Yamakuni T., Tanaka I., Shimizu S., Ikenaka K., Imoto K. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor. J Biol Chem. 1999 Sep 24;274(39):27359–27370. doi: 10.1074/jbc.274.39.27359. [DOI] [PubMed] [Google Scholar]
  34. Osipenko O. N., Barrie A. P., Allen J. M., Gurney A. M. Pituitary adenylyl cyclase-activating peptide activates multiple intracellular signaling pathways to regulate ion channels in PC12 cells. J Biol Chem. 2000 Jun 2;275(22):16626–16631. doi: 10.1074/jbc.M909636199. [DOI] [PubMed] [Google Scholar]
  35. Parekh A. B., Penner R. Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7907–7911. doi: 10.1073/pnas.92.17.7907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Partridge L. D., Valenzuela C. F. Block of hippocampal CAN channels by flufenamate. Brain Res. 2000 Jun 9;867(1-2):143–148. doi: 10.1016/s0006-8993(00)02275-7. [DOI] [PubMed] [Google Scholar]
  37. Petersen C. C., Berridge M. J., Borgese M. F., Bennett D. L. Putative capacitative calcium entry channels: expression of Drosophila trp and evidence for the existence of vertebrate homologues. Biochem J. 1995 Oct 1;311(Pt 1):41–44. doi: 10.1042/bj3110041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Preuss K. D., Nöller J. K., Krause E., Göbel A., Schulz I. Expression and characterization of a trpl homolog from rat. Biochem Biophys Res Commun. 1997 Nov 7;240(1):167–172. doi: 10.1006/bbrc.1997.7528. [DOI] [PubMed] [Google Scholar]
  39. Putney J. W., Jr TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry. Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):14669–14671. doi: 10.1073/pnas.96.26.14669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shi L., Wang C. a. Inhibitory effect of the kinase inhibitor chelerythrine on acetylcholine-induced current in PC12 cells. Arch Biochem Biophys. 1999 Aug 1;368(1):40–44. doi: 10.1006/abbi.1999.1235. [DOI] [PubMed] [Google Scholar]
  41. Sun M., Goldin E., Stahl S., Falardeau J. L., Kennedy J. C., Acierno J. S., Jr, Bove C., Kaneski C. R., Nagle J., Bromley M. C. Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel. Hum Mol Genet. 2000 Oct 12;9(17):2471–2478. doi: 10.1093/hmg/9.17.2471. [DOI] [PubMed] [Google Scholar]
  42. Sutherland C. A., Amin D. Relative activities of rat and dog platelet phospholipase A2 and diglyceride lipase. Selective inhibition of diglyceride lipase by RHC 80267. J Biol Chem. 1982 Dec 10;257(23):14006–14010. [PubMed] [Google Scholar]
  43. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  44. Taylor S. C., Peers C. Store-operated Ca2+ influx and voltage-gated Ca2+ channels coupled to exocytosis in pheochromocytoma (PC12) cells. J Neurochem. 1999 Aug;73(2):874–880. doi: 10.1046/j.1471-4159.1999.0730874.x. [DOI] [PubMed] [Google Scholar]
  45. Zhang L., Saffen D. Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization. J Biol Chem. 2001 Jan 12;276(16):13331–13339. doi: 10.1074/jbc.M008914200. [DOI] [PubMed] [Google Scholar]
  46. 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. doi: 10.1074/jbc.273.1.133. [DOI] [PubMed] [Google Scholar]
  47. Zhu X., Jiang M., Peyton M., Boulay G., Hurst R., Stefani E., Birnbaumer L. trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell. 1996 May 31;85(5):661–671. doi: 10.1016/s0092-8674(00)81233-7. [DOI] [PubMed] [Google Scholar]
  48. Zitt C., Obukhov A. G., Strübing C., Zobel A., Kalkbrenner F., Lückhoff A., Schultz G. Expression of TRPC3 in Chinese hamster ovary cells results in calcium-activated cation currents not related to store depletion. J Cell Biol. 1997 Sep 22;138(6):1333–1341. doi: 10.1083/jcb.138.6.1333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. de Chaffoy de Courcelles D. C., Roevens P., Van Belle H. R 59 022, a diacylglycerol kinase inhibitor. Its effect on diacylglycerol and thrombin-induced C kinase activation in the intact platelet. J Biol Chem. 1985 Dec 15;260(29):15762–15770. [PubMed] [Google Scholar]

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