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 Geiger

Research output: Contribution to journalArticle

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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

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Ryanodine Receptor Calcium Release Channel
HEK293 Cells
Caffeine
Kidney
Ryanodine
Amyloid beta-Protein Precursor
RNA-Directed DNA Polymerase
Cell Lineage
Fibroblasts
Calcium Channels
Reverse Transcriptase Polymerase Chain Reaction
Immunoprecipitation
Amyloid
Polyacrylamide Gel Electrophoresis
Alzheimer Disease
Cells
Calcium
Imaging techniques
Cell Line
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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

Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. / Querfurth, Henry W.; Haughey, Norman J.; Greenway, Steven C.; Yacono, Patrick W.; Golan, David E.; Geiger, Jonathan.

In: Biochemical Journal, Vol. 334, No. 1, 15.08.1998, p. 79-86.

Research output: Contribution to journalArticle

Querfurth, HW, Haughey, NJ, Greenway, SC, Yacono, PW, Golan, DE & Geiger, J 1998, 'Expression of ryanodine receptors in human embryonic kidney (HEK293) cells', Biochemical Journal, vol. 334, no. 1, pp. 79-86. https://doi.org/10.1042/bj3340079
Querfurth, Henry W. ; Haughey, Norman J. ; Greenway, Steven C. ; Yacono, Patrick W. ; Golan, David E. ; Geiger, Jonathan. / Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. In: Biochemical Journal. 1998 ; Vol. 334, No. 1. pp. 79-86.
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