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

Henry W. Querfurth, Norman J. Haughey, Steven C. Greenway, Patrick W. Yacono, David E. Golan, Jonathan D. Geiger

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+(i)) stores increased the release of amyloid β-peptide (Aβ) from transfected human embryonic kidney cells (HEK293). The present study was to test the hypothesis that the caffeine/Aβ 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 (K(d) = 9.9 ± 1.6 nM, B(max) = 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 β-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.

Original languageEnglish (US)
Pages (from-to)79-86
Number of pages8
JournalBiochemical Journal
Volume334
Issue number1
DOIs
StatePublished - Aug 15 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Querfurth, H. W., Haughey, N. J., Greenway, S. C., Yacono, P. W., Golan, D. E., & Geiger, J. D. (1998). Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. Biochemical Journal, 334(1), 79-86. https://doi.org/10.1042/bj3340079