Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Nov 1;131(3):655–667. doi: 10.1083/jcb.131.3.655

Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels

PMCID: PMC2120614  PMID: 7593187

Abstract

Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line NZdipA to explore the molecular mechanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell lines (CJ-1 through CJ-5) were selected based on their failure to express a lethal diphtheria toxin A chain gene and a lacZ reporter gene driven by NF-AT, a Ca(2+)- and protein kinase C-dependent transcription factor. The rate of Ca2+ influx evoked by thapsigargin was reduced to varying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+]i was unaltered, suggesting that the transcriptional defect in these cells is caused by a reduced level of capacitative Ca2+ entry. We examined several factors that determine the rate of Ca2+ entry, including CRAC channel activity, K(+)-channel activity, and Ca2+ clearance mechanisms. The only parameter found to be dramatically altered in most of the mutant lines was the amplitude of the Ca2+ current (ICRAC), which ranged from 1 to 41% of that seen in parental control cells. In each case, the severity of the ICRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca(2-)-dependent gene transcription. Behavior of the mutant cells provides genetic evidence for several roles of ICRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+]i primarily by activating CRAC channels. Second, ICRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx that underlies antigen-mediated T cell activation. These mutant cell lines may provide a useful system for isolating, expressing, and exploring the functions of genes involved in capacitative Ca2+ entry.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Balasubramanyam M., Rohowsky-Kochan C., Reeves J. P., Gardner J. P. Na+/Ca2+ exchange-mediated calcium entry in human lymphocytes. J Clin Invest. 1994 Nov;94(5):2002–2008. doi: 10.1172/JCI117553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cahalan M. D., Chandy K. G., DeCoursey T. E., Gupta S. A voltage-gated potassium channel in human T lymphocytes. J Physiol. 1985 Jan;358:197–237. doi: 10.1113/jphysiol.1985.sp015548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chow S. C., Kass G. E., Orrenius S. Two independently regulated Ca2+ entry mechanisms coexist in Jurkat T cells during T cell receptor antigen activation. Biochem J. 1993 Jul 15;293(Pt 2):395–398. doi: 10.1042/bj2930395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chung S. C., McDonald T. V., Gardner P. Inhibition by SK&F 96365 of Ca2+ current, IL-2 production and activation in T lymphocytes. Br J Pharmacol. 1994 Nov;113(3):861–868. doi: 10.1111/j.1476-5381.1994.tb17072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chused T. M., Wilson H. A., Greenblatt D., Ishida Y., Edison L. J., Tsien R. Y., Finkelman F. D. Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti-IgM and anti-IgD. Cytometry. 1987 Jul;8(4):396–404. doi: 10.1002/cyto.990080409. [DOI] [PubMed] [Google Scholar]
  6. Clipstone N. A., Crabtree G. R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992 Jun 25;357(6380):695–697. doi: 10.1038/357695a0. [DOI] [PubMed] [Google Scholar]
  7. Crabtree G. R., Clipstone N. A. Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu Rev Biochem. 1994;63:1045–1083. doi: 10.1146/annurev.bi.63.070194.005145. [DOI] [PubMed] [Google Scholar]
  8. Densmore J. J., Szabo G., Gray L. S. A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes. FEBS Lett. 1992 Nov 9;312(2-3):161–164. doi: 10.1016/0014-5793(92)80926-8. [DOI] [PubMed] [Google Scholar]
  9. Dolmetsch R. E., Lewis R. S. Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes. J Gen Physiol. 1994 Mar;103(3):365–388. doi: 10.1085/jgp.103.3.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donnadieu E., Bismuth G., Trautmann A. Calcium fluxes in T lymphocytes. J Biol Chem. 1992 Dec 25;267(36):25864–25872. [PubMed] [Google Scholar]
  11. Dupuis G., Héroux J., Payet M. D. Characterization of Ca2+ and K+ currents in the human Jurkat T cell line: effects of phytohaemagglutinin. J Physiol. 1989 May;412:135–154. doi: 10.1113/jphysiol.1989.sp017608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Durand D. B., Shaw J. P., Bush M. R., Replogle R. E., Belagaje R., Crabtree G. R. Characterization of antigen receptor response elements within the interleukin-2 enhancer. Mol Cell Biol. 1988 Apr;8(4):1715–1724. doi: 10.1128/mcb.8.4.1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fasolato C., Hoth M., Matthews G., Penner R. Ca2+ and Mn2+ influx through receptor-mediated activation of nonspecific cation channels in mast cells. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):3068–3072. doi: 10.1073/pnas.90.7.3068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fasolato C., Innocenti B., Pozzan T. Receptor-activated Ca2+ influx: how many mechanisms for how many channels? Trends Pharmacol Sci. 1994 Mar;15(3):77–83. doi: 10.1016/0165-6147(94)90282-8. [DOI] [PubMed] [Google Scholar]
  15. Fiering S., Northrop J. P., Nolan G. P., Mattila P. S., Crabtree G. R., Herzenberg L. A. Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 1990 Oct;4(10):1823–1834. doi: 10.1101/gad.4.10.1823. [DOI] [PubMed] [Google Scholar]
  16. Flanagan W. M., Corthésy B., Bram R. J., Crabtree G. R. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature. 1991 Aug 29;352(6338):803–807. doi: 10.1038/352803a0. [DOI] [PubMed] [Google Scholar]
  17. Franzius D., Hoth M., Penner R. Non-specific effects of calcium entry antagonists in mast cells. Pflugers Arch. 1994 Oct;428(5-6):433–438. doi: 10.1007/BF00374562. [DOI] [PubMed] [Google Scholar]
  18. Ghosh T. K., Bian J. H., Short A. D., Rybak S. L., Gill D. L. Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth. J Biol Chem. 1991 Dec 25;266(36):24690–24697. [PubMed] [Google Scholar]
  19. Goldsmith M. A., Dazin P. F., Weiss A. At least two non-antigen-binding molecules are required for signal transduction by the T-cell antigen receptor. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8613–8617. doi: 10.1073/pnas.85.22.8613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldsmith M. A., Weiss A. Early signal transduction by the antigen receptor without commitment to T cell activation. Science. 1988 May 20;240(4855):1029–1031. doi: 10.1126/science.3259335. [DOI] [PubMed] [Google Scholar]
  21. Gouy H., Cefai D., Christensen S. B., Debré P., Bismuth G. Ca2+ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca2+ stores. A study with the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin. Eur J Immunol. 1990 Oct;20(10):2269–2275. doi: 10.1002/eji.1830201016. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hess S. D., Oortgiesen M., Cahalan M. D. Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx. J Immunol. 1993 Apr 1;150(7):2620–2633. [PubMed] [Google Scholar]
  24. Hoth M., Penner R. Calcium release-activated calcium current in rat mast cells. J Physiol. 1993 Jun;465:359–386. doi: 10.1113/jphysiol.1993.sp019681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Jacob R. Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells. J Physiol. 1990 Feb;421:55–77. doi: 10.1113/jphysiol.1990.sp017933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kao F. T., Puck T. T. Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents. J Cell Physiol. 1969 Dec;74(3):245–258. doi: 10.1002/jcp.1040740305. [DOI] [PubMed] [Google Scholar]
  28. Khan A. A., Steiner J. P., Klein M. G., Schneider M. F., Snyder S. H. IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor. Science. 1992 Aug 7;257(5071):815–818. doi: 10.1126/science.1323146. [DOI] [PubMed] [Google Scholar]
  29. Kuno M., Gardner P. Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes. Nature. 1987 Mar 19;326(6110):301–304. doi: 10.1038/326301a0. [DOI] [PubMed] [Google Scholar]
  30. Lewis R. S., Cahalan M. D. Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells. Cell Regul. 1989 Nov;1(1):99–112. doi: 10.1091/mbc.1.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lewis R. S., Cahalan M. D. Potassium and calcium channels in lymphocytes. Annu Rev Immunol. 1995;13:623–653. doi: 10.1146/annurev.iy.13.040195.003203. [DOI] [PubMed] [Google Scholar]
  32. Lin C. S., Boltz R. C., Blake J. T., Nguyen M., Talento A., Fischer P. A., Springer M. S., Sigal N. H., Slaughter R. S., Garcia M. L. Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation. J Exp Med. 1993 Mar 1;177(3):637–645. doi: 10.1084/jem.177.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Low A. M., Kwan C. Y., Daniel E. E. Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms. Am J Physiol. 1992 Jan;262(1 Pt 2):H31–H37. doi: 10.1152/ajpheart.1992.262.1.H31. [DOI] [PubMed] [Google Scholar]
  34. Lückhoff A., Clapham D. E. Calcium channels activated by depletion of internal calcium stores in A431 cells. Biophys J. 1994 Jul;67(1):177–182. doi: 10.1016/S0006-3495(94)80467-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mason M. J., Grinstein S. Ionomycin activates electrogenic Ca2+ influx in rat thymic lymphocytes. Biochem J. 1993 Nov 15;296(Pt 1):33–39. doi: 10.1042/bj2960033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mason M. J., Mahaut-Smith M. P., Grinstein S. The role of intracellular Ca2+ in the regulation of the plasma membrane Ca2+ permeability of unstimulated rat lymphocytes. J Biol Chem. 1991 Jun 15;266(17):10872–10879. [PubMed] [Google Scholar]
  37. Montero M., Alvarez J., García-Sancho J. Control of plasma-membrane Ca2+ entry by the intracellular Ca2+ stores. Kinetic evidence for a short-lived mediator. Biochem J. 1992 Dec 1;288(Pt 2):519–525. doi: 10.1042/bj2880519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Morgan A. J., Jacob R. Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane. Biochem J. 1994 Jun 15;300(Pt 3):665–672. doi: 10.1042/bj3000665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nakanishi T., Kohno K., Ishiura M., Ohashi H., Uchida T. Complete nucleotide sequence and characterization of the 5'-flanking region of mammalian elongation factor 2 gene. J Biol Chem. 1988 May 5;263(13):6384–6391. [PubMed] [Google Scholar]
  40. Negulescu P. A., Shastri N., Cahalan M. D. Intracellular calcium dependence of gene expression in single T lymphocytes. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2873–2877. doi: 10.1073/pnas.91.7.2873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Partiseti M., Le Deist F., Hivroz C., Fischer A., Korn H., Choquet D. The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency. J Biol Chem. 1994 Dec 23;269(51):32327–32335. [PubMed] [Google Scholar]
  42. Penner R., Fasolato C., Hoth M. Calcium influx and its control by calcium release. Curr Opin Neurobiol. 1993 Jun;3(3):368–374. doi: 10.1016/0959-4388(93)90130-q. [DOI] [PubMed] [Google Scholar]
  43. Premack B. A., McDonald T. V., Gardner P. Activation of Ca2+ current in Jurkat T cells following the depletion of Ca2+ stores by microsomal Ca(2+)-ATPase inhibitors. J Immunol. 1994 Jun 1;152(11):5226–5240. [PubMed] [Google Scholar]
  44. Preston S. F., Sha'afi R. I., Berlin R. D. Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis. Cell Regul. 1991 Nov;2(11):915–925. doi: 10.1091/mbc.2.11.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Putney J. W., Jr Capacitative calcium entry revisited. Cell Calcium. 1990 Nov-Dec;11(10):611–624. doi: 10.1016/0143-4160(90)90016-n. [DOI] [PubMed] [Google Scholar]
  47. Rao A. NF-ATp: a transcription factor required for the co-ordinate induction of several cytokine genes. Immunol Today. 1994 Jun;15(6):274–281. doi: 10.1016/0167-5699(94)90007-8. [DOI] [PubMed] [Google Scholar]
  48. Sarkadi B., Tordai A., Homolya L., Scharff O., Gárdos G. Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts. J Membr Biol. 1991 Jul;123(1):9–21. doi: 10.1007/BF01993958. [DOI] [PubMed] [Google Scholar]
  49. Sei Y., Takemura M., Gusovsky F., Skolnick P., Basile A. Distinct mechanisms for Ca2+ entry induced by OKT3 and Ca2+ depletion in Jurkat T cells. Exp Cell Res. 1995 Jan;216(1):222–231. doi: 10.1006/excr.1995.1028. [DOI] [PubMed] [Google Scholar]
  50. Serafini A. T., Lewis R. S., Clipstone N. A., Bram R. J., Fanger C., Fiering S., Herzenberg L. A., Crabtree G. R. Isolation of mutant T lymphocytes with defects in capacitative calcium entry. Immunity. 1995 Aug;3(2):239–250. doi: 10.1016/1074-7613(95)90093-4. [DOI] [PubMed] [Google Scholar]
  51. Stemmer P. M., Klee C. B. Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B. Biochemistry. 1994 Jun 7;33(22):6859–6866. doi: 10.1021/bi00188a015. [DOI] [PubMed] [Google Scholar]
  52. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Truneh A., Albert F., Golstein P., Schmitt-Verhulst A. M. Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester. Nature. 1985 Jan 24;313(6000):318–320. doi: 10.1038/313318a0. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Villalobos C., Fonteriz R., López M. G., García A. G., García-Sancho J. Inhibition of voltage-gated Ca2+ entry into GH3 and chromaffin cells by imidazole antimycotics and other cytochrome P450 blockers. FASEB J. 1992 Jun;6(9):2742–2747. doi: 10.1096/fasebj.6.9.1319362. [DOI] [PubMed] [Google Scholar]
  56. Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
  57. Wilson O. I., Marriott I., Mahaut-Smith M. P., Hymel L. J., Mason M. J. Isolation and characterization of membrane potential changes associated with release of calcium from intracellular stores in rat thymic lymphocytes. J Membr Biol. 1994 Jan;137(2):159–168. doi: 10.1007/BF00233485. [DOI] [PubMed] [Google Scholar]
  58. Zweifach A., Lewis R. S. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6295–6299. doi: 10.1073/pnas.90.13.6295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zweifach A., Lewis R. S. Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback. J Gen Physiol. 1995 Feb;105(2):209–226. doi: 10.1085/jgp.105.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zweifach A., Lewis R. S. Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms. J Biol Chem. 1995 Jun 16;270(24):14445–14451. doi: 10.1074/jbc.270.24.14445. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES