Erythropoietin modulates calcium influx through TRPC2

Xin Chu, Joseph Y. Cheung, Dwayne L. Barber, Lutz Birnbaumer, Lawrence I. Rothblum, Kathleen Conrad, Virginia Abrasonis, Yiu Mo Chan, Richard Stahl, David J. Carey, Barbara Miller

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

Mammalian isoforms of calcium-permeable Drosophila transient receptor potential channels (TRPC) are involved in the sustained phase of calcium entry in non-excitable cells. Erythropoietin (Epo) stimulates a rise in intracellular calcium ([Ca]i) via activation of voltage-independent calcium channel(s) in erythroid cells. Here, involvement of murine orthologs of classical TRPC in the Epo-modulated increase in [Ca]i was examined. RT-PCR of TRPC 1-6 revealed high expression of only TRPC2 in Epo-dependent cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca]i. Using RT-PCR, Western blotting, and immunolocalization, expression of the longest isoform of mTRPC2, clone 14, was demonstrated in HCD-57 cells, Ba/F3 Epo-R cells, and primary murine erythroblasts. To determine whether erythropoietin is capable of modulating calcium influx through TRPC2, CHO cells were cotransfected with Epo-R subcloned into pTracer-CMV and either murine TRPC2 clone 14 or TRPC6, a negative control, into pQBI50. Successful transfection of Epo-R was verified in single cells by detection of green fluorescent protein from pTracer-CMV using digital video imaging, and successful transfection of TRPC was confirmed by detection of blue fluorescent protein fused through a flexible linker to TRPC. [Ca]i changes were simultaneously monitored in cells loaded with Rhod-2 or Fura Red. Epo stimulation of CHO cells cotransfected with Epo-R and TRPC2 resulted in a rise in [Ca]i above base line (372 ± 71%), which was significantly greater (p ≤ 0.0007) than that seen in cells transfected with TRPC6 or empty pQBI50 vector. This rise in [Ca]i required Epo and extracellular calcium. These results identify a calcium-permeable channel, TRPC2, in erythroid cells and demonstrate modulation of calcium influx through this channel by erythropoietin.

Original languageEnglish (US)
Pages (from-to)34375-34382
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number37
DOIs
StatePublished - Sep 13 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Chu, X., Cheung, J. Y., Barber, D. L., Birnbaumer, L., Rothblum, L. I., Conrad, K., Abrasonis, V., Chan, Y. M., Stahl, R., Carey, D. J., & Miller, B. (2002). Erythropoietin modulates calcium influx through TRPC2. Journal of Biological Chemistry, 277(37), 34375-34382. https://doi.org/10.1074/jbc.M205541200