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. 1998 Aug 15;334(Pt 1):79–86. doi: 10.1042/bj3340079

Expression of ryanodine receptors in human embryonic kidney (HEK293) cells.

H W Querfurth 1, N J Haughey 1, S C Greenway 1, P W Yacono 1, D E Golan 1, J D Geiger 1
PMCID: PMC1219664  PMID: 9693105

Abstract

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid beta-peptide (Abeta) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580-1591]. The present study was to test the hypothesis that the caffeine/Abeta responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd=9.9+/-1.6 nM, Bmax=25+/-4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5-15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the beta-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.

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Selected References

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  1. Airey J. A., Baring M. D., Sutko J. L. Ryanodine receptor protein is expressed during differentiation in the muscle cell lines BC3H1 and C2C12. Dev Biol. 1991 Nov;148(1):365–374. doi: 10.1016/0012-1606(91)90344-3. [DOI] [PubMed] [Google Scholar]
  2. Airey J. A., Grinsell M. M., Jones L. R., Sutko J. L., Witcher D. Three ryanodine receptor isoforms exist in avian striated muscles. Biochemistry. 1993 Jun 8;32(22):5739–5745. doi: 10.1021/bi00073a003. [DOI] [PubMed] [Google Scholar]
  3. Anderson C. M., Xiong W., Young J. D., Cass C. E., Parkinson F. E. Demonstration of the existence of mRNAs encoding N1/cif and N2/cit sodium/nucleoside cotransporters in rat brain. Brain Res Mol Brain Res. 1996 Dec;42(2):358–361. doi: 10.1016/s0169-328x(96)00244-6. [DOI] [PubMed] [Google Scholar]
  4. Bennett D. L., Cheek T. R., Berridge M. J., De Smedt H., Parys J. B., Missiaen L., Bootman M. D. Expression and function of ryanodine receptors in nonexcitable cells. J Biol Chem. 1996 Mar 15;271(11):6356–6362. doi: 10.1074/jbc.271.11.6356. [DOI] [PubMed] [Google Scholar]
  5. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  6. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  7. Campbell K. P., Knudson C. M., Imagawa T., Leung A. T., Sutko J. L., Kahl S. D., Raab C. R., Madson L. Identification and characterization of the high affinity [3H]ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel. J Biol Chem. 1987 May 15;262(14):6460–6463. [PubMed] [Google Scholar]
  8. Cooper D. M., Mons N., Karpen J. W. Adenylyl cyclases and the interaction between calcium and cAMP signalling. Nature. 1995 Mar 30;374(6521):421–424. doi: 10.1038/374421a0. [DOI] [PubMed] [Google Scholar]
  9. Ellisman M. H., Deerinck T. J., Ouyang Y., Beck C. F., Tanksley S. J., Walton P. D., Airey J. A., Sutko J. L. Identification and localization of ryanodine binding proteins in the avian central nervous system. Neuron. 1990 Aug;5(2):135–146. doi: 10.1016/0896-6273(90)90304-x. [DOI] [PubMed] [Google Scholar]
  10. Felder C. C., Poulter M. O., Wess J. Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellular Ca2+. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):509–513. doi: 10.1073/pnas.89.2.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fleischer S., Inui M. Biochemistry and biophysics of excitation-contraction coupling. Annu Rev Biophys Biophys Chem. 1989;18:333–364. doi: 10.1146/annurev.bb.18.060189.002001. [DOI] [PubMed] [Google Scholar]
  12. Flucher B. E. Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad. Dev Biol. 1992 Dec;154(2):245–260. doi: 10.1016/0012-1606(92)90065-o. [DOI] [PubMed] [Google Scholar]
  13. Giannini G., Clementi E., Ceci R., Marziali G., Sorrentino V. Expression of a ryanodine receptor-Ca2+ channel that is regulated by TGF-beta. Science. 1992 Jul 3;257(5066):91–94. doi: 10.1126/science.1320290. [DOI] [PubMed] [Google Scholar]
  14. Giannini G., Conti A., Mammarella S., Scrobogna M., Sorrentino V. The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues. J Cell Biol. 1995 Mar;128(5):893–904. doi: 10.1083/jcb.128.5.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnson R. M., King K. L., Morhenn V. B. Comparison of second messenger formation in human keratinocytes following stimulation with epidermal growth factor and bradykinin. Second Messengers Phosphoproteins. 1992;14(1-2):21–37. [PubMed] [Google Scholar]
  16. Kijima Y., Saito A., Jetton T. L., Magnuson M. A., Fleischer S. Different intracellular localization of inositol 1,4,5-trisphosphate and ryanodine receptors in cardiomyocytes. J Biol Chem. 1993 Feb 15;268(5):3499–3506. [PubMed] [Google Scholar]
  17. Lee H. C., Walseth T. F., Bratt G. T., Hayes R. N., Clapper D. L. Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity. J Biol Chem. 1989 Jan 25;264(3):1608–1615. [PubMed] [Google Scholar]
  18. McNulty T. J., Taylor C. W. Caffeine-stimulated Ca2+ release from the intracellular stores of hepatocytes is not mediated by ryanodine receptors. Biochem J. 1993 May 1;291(Pt 3):799–801. doi: 10.1042/bj2910799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McPherson P. S., Campbell K. P. Characterization of the major brain form of the ryanodine receptor/Ca2+ release channel. J Biol Chem. 1993 Sep 15;268(26):19785–19790. [PubMed] [Google Scholar]
  20. McPherson P. S., Campbell K. P. The ryanodine receptor/Ca2+ release channel. J Biol Chem. 1993 Jul 5;268(19):13765–13768. [PubMed] [Google Scholar]
  21. Meissner G. Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum. J Biol Chem. 1986 May 15;261(14):6300–6306. [PubMed] [Google Scholar]
  22. Meissner G. Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annu Rev Physiol. 1994;56:485–508. doi: 10.1146/annurev.ph.56.030194.002413. [DOI] [PubMed] [Google Scholar]
  23. Merle P. L., Feige J. J., Verdetti J. Basic fibroblast growth factor activates calcium channels in neonatal rat cardiomyocytes. J Biol Chem. 1995 Jul 21;270(29):17361–17367. doi: 10.1074/jbc.270.29.17361. [DOI] [PubMed] [Google Scholar]
  24. Ogawa Y. Role of ryanodine receptors. Crit Rev Biochem Mol Biol. 1994;29(4):229–274. doi: 10.3109/10409239409083482. [DOI] [PubMed] [Google Scholar]
  25. Padua R. A., Nagy J. I., Geiger J. D. Ionic strength dependence of calcium, adenine nucleotide, magnesium, and caffeine actions on ryanodine receptors in rat brain. J Neurochem. 1994 Jun;62(6):2340–2348. doi: 10.1046/j.1471-4159.1994.62062340.x. [DOI] [PubMed] [Google Scholar]
  26. Pritchett D. B., Sontheimer H., Gorman C. M., Kettenmann H., Seeburg P. H., Schofield P. R. Transient expression shows ligand gating and allosteric potentiation of GABAA receptor subunits. Science. 1988 Dec 2;242(4883):1306–1308. doi: 10.1126/science.2848320. [DOI] [PubMed] [Google Scholar]
  27. Putney J. W., Jr Excitement about calcium signaling in inexcitable cells. Science. 1993 Oct 29;262(5134):676–678. doi: 10.1126/science.8235587. [DOI] [PubMed] [Google Scholar]
  28. Qiu W. Q., Ferreira A., Miller C., Koo E. H., Selkoe D. J. Cell-surface beta-amyloid precursor protein stimulates neurite outgrowth of hippocampal neurons in an isoform-dependent manner. J Neurosci. 1995 Mar;15(3 Pt 2):2157–2167. doi: 10.1523/JNEUROSCI.15-03-02157.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Querfurth H. W., Jiang J., Geiger J. D., Selkoe D. J. Caffeine stimulates amyloid beta-peptide release from beta-amyloid precursor protein-transfected HEK293 cells. J Neurochem. 1997 Oct;69(4):1580–1591. doi: 10.1046/j.1471-4159.1997.69041580.x. [DOI] [PubMed] [Google Scholar]
  30. Simpson P. B., Challiss R. A., Nahorski S. R. Neuronal Ca2+ stores: activation and function. Trends Neurosci. 1995 Jul;18(7):299–306. doi: 10.1016/0166-2236(95)93919-o. [DOI] [PubMed] [Google Scholar]
  31. Socci R., Chu A., Reinach P., Mészáros L. G. In situ Ca(2+)-induced Ca2+ release from a ryanodine-sensitive intracellular Ca2+ store in corneal epithelial cells. Comp Biochem Physiol B. 1993 Dec;106(4):793–797. doi: 10.1016/0305-0491(93)90032-z. [DOI] [PubMed] [Google Scholar]
  32. Stein M. B., Padua R. A., Nagy J. I., Geiger J. D. High affinity [3H]ryanodine binding sites in postmortem human brain: regional distribution and effects of calcium, magnesium and caffeine. Brain Res. 1992 Jul 10;585(1-2):349–354. doi: 10.1016/0006-8993(92)91235-7. [DOI] [PubMed] [Google Scholar]
  33. Toyofuku T., Yabuki M., Otsu K., Kuzuya T., Hori M., Tada M. Intercellular calcium signaling via gap junction in connexin-43-transfected cells. J Biol Chem. 1998 Jan 16;273(3):1519–1528. doi: 10.1074/jbc.273.3.1519. [DOI] [PubMed] [Google Scholar]
  34. Tunwell R. E., Lai F. A. Ryanodine receptor expression in the kidney and a non-excitable kidney epithelial cell. J Biol Chem. 1996 Nov 22;271(47):29583–29588. doi: 10.1074/jbc.271.47.29583. [DOI] [PubMed] [Google Scholar]
  35. Volpe P., Villa A., Damiani E., Sharp A. H., Podini P., Snyder S. H., Meldolesi J. Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons. EMBO J. 1991 Nov;10(11):3183–3189. doi: 10.1002/j.1460-2075.1991.tb04880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wagner K. A., Yacono P. W., Golan D. E., Tashjian A. H., Jr Mechanism of spontaneous intracellular calcium fluctuations in single GH4C1 rat pituitary cells. Biochem J. 1993 May 15;292(Pt 1):175–182. doi: 10.1042/bj2920175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zacchetti D., Clementi E., Fasolato C., Lorenzon P., Zottini M., Grohovaz F., Fumagalli G., Pozzan T., Meldolesi J. Intracellular Ca2+ pools in PC12 cells. A unique, rapidly exchanging pool is sensitive to both inositol 1,4,5-trisphosphate and caffeine-ryanodine. J Biol Chem. 1991 Oct 25;266(30):20152–20158. [PubMed] [Google Scholar]
  38. Zhao Y., Sudol M., Hanafusa H., Krueger J. Increased tyrosine kinase activity of c-Src during calcium-induced keratinocyte differentiation. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8298–8302. doi: 10.1073/pnas.89.17.8298. [DOI] [PMC free article] [PubMed] [Google Scholar]

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