Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Apr 1;331(Pt 1):331–339. doi: 10.1042/bj3310331

Functional expression of TrpC1: a human homologue of the Drosophila Trp channel.

W G Sinkins 1, M Estacion 1, W P Schilling 1
PMCID: PMC1219356  PMID: 9512497

Abstract

TrpC1 appears to be a store-operated channel (SOC) when expressed in mammalian cells. In the present study, TrpC1 was expressed in Sf9 insect cells using the baculovirus expression system. Expression of TrpC1 caused an increase in basal cytosolic free Ca2+ concentration ([Ca2+]i) as a function of post-infection time. Basal Ba2+ influx, an index of plasmalemmal Ca2+ permeability, was also increased and was blocked by La3+. Although the thapsigargin-induced change in [Ca2+]i was greater in TrpC1-expressing cells than controls, Ba2+ influx was unaffected by thapsigargin. Whole-cell membrane currents recorded in TrpC1-expressing cells increased as a function of post-infection time and were (1) inwardly rectifying in symmetrical sodium gluconate solutions, (2) non-selective with respect to Na+, Ca2+ and Ba2+, and (3) blocked by La3+. Furthermore TrpC1 currents were unaffected by (1) thapsigargin, (2) dialysis of the cell with Ins(1,4,5)P3 or (3) dialysis of the cell with solutions containing high concentrations of the Ca2+ chelator, EGTA. These results suggest that TrpC1 forms non-selective cation channels that are constitutively active when expressed in Sf9 cells, but insensitive to depletion of the internal Ca2+ stores. Thus TrpC1 may be a subunit of a SOC which alone can form functional channels in Sf9 cells, but which requires additional subunits or cytoplasmic factors present in mammalian cells for expression of SOC activity.

Full Text

The Full Text of this article is available as a PDF (779.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Chang A. S., Chang S. M., Garcia R. L., Schilling W. P. Concomitant and hormonally regulated expression of trp genes in bovine aortic endothelial cells. FEBS Lett. 1997 Oct 6;415(3):335–340. doi: 10.1016/s0014-5793(97)01155-1. [DOI] [PubMed] [Google Scholar]
  3. Chevesich J., Kreuz A. J., Montell C. Requirement for the PDZ domain protein, INAD, for localization of the TRP store-operated channel to a signaling complex. Neuron. 1997 Jan;18(1):95–105. doi: 10.1016/s0896-6273(01)80049-0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Gomez-Lagunas F., Armstrong C. M. Inactivation in ShakerB K+ channels: a test for the number of inactivating particles on each channel. Biophys J. 1995 Jan;68(1):89–95. doi: 10.1016/S0006-3495(95)80162-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  7. Gubitosi-Klug R. A., Gross R. W. Fatty acid ethyl esters, nonoxidative metabolites of ethanol, accelerate the kinetics of activation of the human brain delayed rectifier K+ channel, Kv1.1. J Biol Chem. 1996 Dec 20;271(51):32519–32522. doi: 10.1074/jbc.271.51.32519. [DOI] [PubMed] [Google Scholar]
  8. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  9. Hardie R. C., Minke B. Phosphoinositide-mediated phototransduction in Drosophila photoreceptors: the role of Ca2+ and trp. Cell Calcium. 1995 Oct;18(4):256–274. doi: 10.1016/0143-4160(95)90023-3. [DOI] [PubMed] [Google Scholar]
  10. Hoth M., Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992 Jan 23;355(6358):353–356. doi: 10.1038/355353a0. [DOI] [PubMed] [Google Scholar]
  11. Hu Y., Schilling W. P. Receptor-mediated activation of recombinant Trpl expressed in Sf9 insect cells. Biochem J. 1995 Jan 15;305(Pt 2):605–611. doi: 10.1042/bj3050605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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] [Google Scholar]
  13. Klaiber K., Williams N., Roberts T. M., Papazian D. M., Jan L. Y., Miller C. Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line. Neuron. 1990 Aug;5(2):221–226. doi: 10.1016/0896-6273(90)90311-3. [DOI] [PubMed] [Google Scholar]
  14. Larsen E. H., Price E. M., Gabriel S. E., Stutts M. J., Boucher R. C. Clusters of Cl- channels in CFTR-expressing Sf9 cells switch spontaneously between slow and fast gating modes. Pflugers Arch. 1996 Jul;432(3):528–537. doi: 10.1007/s004240050166. [DOI] [PubMed] [Google Scholar]
  15. Marks AR. Immunophilin Modulation of Calcium Channel Gating. Methods. 1996 Apr;9(2):177–187. doi: 10.1006/meth.1996.0024. [DOI] [PubMed] [Google Scholar]
  16. Marten I., Gaymard F., Lemaillet G., Thibaud J. B., Sentenac H., Hedrich R. Functional expression of the plant K+ channel KAT1 in insect cells. FEBS Lett. 1996 Feb 19;380(3):229–232. doi: 10.1016/0014-5793(96)00042-7. [DOI] [PubMed] [Google Scholar]
  17. Obukhov A. G., Harteneck C., Zobel A., Harhammer R., Kalkbrenner F., Leopoldt D., Lückhoff A., Nürnberg B., Schultz G. Direct activation of trpl cation channels by G alpha11 subunits. EMBO J. 1996 Nov 1;15(21):5833–5838. [PMC free article] [PubMed] [Google Scholar]
  18. Parekh A. B., Penner R. Store depletion and calcium influx. Physiol Rev. 1997 Oct;77(4):901–930. doi: 10.1152/physrev.1997.77.4.901. [DOI] [PubMed] [Google Scholar]
  19. Philipp S., Cavalié A., Freichel M., Wissenbach U., Zimmer S., Trost C., Marquart A., Murakami M., Flockerzi V. A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J. 1996 Nov 15;15(22):6166–6171. [PMC free article] [PubMed] [Google Scholar]
  20. Puri T. S., Gerhardstein B. L., Zhao X. L., Ladner M. B., Hosey M. M. Differential effects of subunit interactions on protein kinase A- and C-mediated phosphorylation of L-type calcium channels. Biochemistry. 1997 Aug 5;36(31):9605–9615. doi: 10.1021/bi970500d. [DOI] [PubMed] [Google Scholar]
  21. Putney J. W., Jr, Bird G. S. The inositol phosphate-calcium signaling system in nonexcitable cells. Endocr Rev. 1993 Oct;14(5):610–631. doi: 10.1210/edrv-14-5-610. [DOI] [PubMed] [Google Scholar]
  22. Pérez-Cornejo P., Begenisich T. The multi-ion nature of the pore in Shaker K+ channels. Biophys J. 1994 Jun;66(6):1929–1938. doi: 10.1016/S0006-3495(94)80986-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Radford K. M., Virginio C., Surprenant A., North R. A., Kawashima E. Baculovirus expression provides direct evidence for heteromeric assembly of P2X2 and P2X3 receptors. J Neurosci. 1997 Sep 1;17(17):6529–6533. doi: 10.1523/JNEUROSCI.17-17-06529.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schilling W. P., Rajan L., Strobl-Jager E. Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics. J Biol Chem. 1989 Aug 5;264(22):12838–12848. [PubMed] [Google Scholar]
  25. Schwalbe R. A., Wang Z., Bianchi L., Brown A. M. Novel sites of N-glycosylation in ROMK1 reveal the putative pore-forming segment H5 as extracellular. J Biol Chem. 1996 Sep 27;271(39):24201–24206. doi: 10.1074/jbc.271.39.24201. [DOI] [PubMed] [Google Scholar]
  26. Schwalbe R. A., Wang Z., Wible B. A., Brown A. M. Potassium channel structure and function as reported by a single glycosylation sequon. J Biol Chem. 1995 Jun 23;270(25):15336–15340. doi: 10.1074/jbc.270.25.15336. [DOI] [PubMed] [Google Scholar]
  27. Shieh B. H., Zhu M. Y. Regulation of the TRP Ca2+ channel by INAD in Drosophila photoreceptors. Neuron. 1996 May;16(5):991–998. doi: 10.1016/s0896-6273(00)80122-1. [DOI] [PubMed] [Google Scholar]
  28. Shuttleworth T. J., Thompson J. L. Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes. J Biol Chem. 1991 Jan 25;266(3):1410–1414. [PubMed] [Google Scholar]
  29. Sinkins W. G., Vaca L., Hu Y., Kunze D. L., Schilling W. P. The COOH-terminal domain of Drosophila TRP channels confers thapsigargin sensitivity. J Biol Chem. 1996 Feb 9;271(6):2955–2960. doi: 10.1074/jbc.271.6.2955. [DOI] [PubMed] [Google Scholar]
  30. Tsunoda S., Sierralta J., Sun Y., Bodner R., Suzuki E., Becker A., Socolich M., Zuker C. S. A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade. Nature. 1997 Jul 17;388(6639):243–249. doi: 10.1038/40805. [DOI] [PubMed] [Google Scholar]
  31. Vaca L., Sinkins W. G., Hu Y., Kunze D. L., Schilling W. P. Activation of recombinant trp by thapsigargin in Sf9 insect cells. Am J Physiol. 1994 Nov;267(5 Pt 1):C1501–C1505. doi: 10.1152/ajpcell.1994.267.5.C1501. [DOI] [PubMed] [Google Scholar]
  32. Wes P. D., Chevesich J., Jeromin A., Rosenberg C., Stetten G., Montell C. TRPC1, a human homolog of a Drosophila store-operated channel. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9652–9656. doi: 10.1073/pnas.92.21.9652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Xu X. Z., Li H. S., Guggino W. B., Montell C. Coassembly of TRP and TRPL produces a distinct store-operated conductance. Cell. 1997 Jun 27;89(7):1155–1164. doi: 10.1016/s0092-8674(00)80302-5. [DOI] [PubMed] [Google Scholar]
  34. Zhu X., Chu P. B., Peyton M., Birnbaumer L. Molecular cloning of a widely expressed human homologue for the Drosophila trp gene. FEBS Lett. 1995 Oct 16;373(3):193–198. doi: 10.1016/0014-5793(95)01038-g. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Zitt C., Zobel A., Obukhov A. G., Harteneck C., Kalkbrenner F., Lückhoff A., Schultz G. Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion. Neuron. 1996 Jun;16(6):1189–1196. doi: 10.1016/s0896-6273(00)80145-2. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES