Interaction of TRPC2 and TRPC6 in Erythropoietin Modulation of Calcium Influx

Xin Chu, Qin Tong, Joseph Y. Cheung, Jocelyn Wozney, Kathleen Conrad, Virginia Mazack, Wenyi Zhang, Richard Stahl, Dwayne L. Barber, Barbara Miller

Research output: Contribution to journalArticle

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Abstract

Erythropoietin (Epo) modulates calcium influx through voltage-independent calcium-permeable channel(s). Here, we characterized the expression of transient receptor potential channels (TRPCs) in primary erythroid cells and examined their regulation. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in [Ca]i. Among the classical TRPC channels, expression of three N-terminal splice variants of TRPC2 (clones 14, 17, and a) and of TRPC6 were demonstrated in these erythroblasts by both reverse transcriptase-PCR and Western blotting. Confocal microscopy confirmed localization to the plasma membrane. To determine the function of individual TRPC channels in erythropoietin modulation of calcium influx, digital video imaging was used to measure calcium influx through these TRPCs in a Chinese hamster ovary (CHO) cell model. Single CHO-S cells, expressing transfected Epo-R, were identified by detection of green fluorescent protein. Cells that express transfected TRPCs were identified by detection of blue fluorescent protein. [Ca]i was monitored with Fura Red. Epo stimulation of CHO-S cells transfected with single TRPC2 isoforms (clone 14, 17, or α) and Epo-R resulted in a significant increase in [Ca]i. This was not observed in cells transfected with Epo-R and TRPC6. In addition, coexpression of TRPC6 with TRPC2 and Epo-R inhibited the increase in [Ca]i observed after Epo stimulation. Immunoprecipitation experiments demonstrated that TRPC2 associates with TRPC6, indicating that these TRPCs can form multimeric channels. These data demonstrate that specific TRPCs are expressed in primary erythroid cells and that two of these channels, TRPC2 and TRPC6, can interact to modulate calcium influx stimulated by erythropoietin.

Original languageEnglish (US)
Pages (from-to)10514-10522
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number11
DOIs
StatePublished - Mar 12 2004

Fingerprint

Erythropoietin
Transient Receptor Potential Channels
Modulation
Calcium
Cricetulus
Ovary
Erythroblasts
Erythroid Cells
Clone Cells
Confocal microscopy
RNA-Directed DNA Polymerase
Calcium Channels
Cell membranes
Green Fluorescent Proteins
Reverse Transcriptase Polymerase Chain Reaction
Immunoprecipitation
Confocal Microscopy
Protein Isoforms
Spleen
Western Blotting

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Chu, Xin ; Tong, Qin ; Cheung, Joseph Y. ; Wozney, Jocelyn ; Conrad, Kathleen ; Mazack, Virginia ; Zhang, Wenyi ; Stahl, Richard ; Barber, Dwayne L. ; Miller, Barbara. / Interaction of TRPC2 and TRPC6 in Erythropoietin Modulation of Calcium Influx. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 11. pp. 10514-10522.
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abstract = "Erythropoietin (Epo) modulates calcium influx through voltage-independent calcium-permeable channel(s). Here, we characterized the expression of transient receptor potential channels (TRPCs) in primary erythroid cells and examined their regulation. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in [Ca]i. Among the classical TRPC channels, expression of three N-terminal splice variants of TRPC2 (clones 14, 17, and a) and of TRPC6 were demonstrated in these erythroblasts by both reverse transcriptase-PCR and Western blotting. Confocal microscopy confirmed localization to the plasma membrane. To determine the function of individual TRPC channels in erythropoietin modulation of calcium influx, digital video imaging was used to measure calcium influx through these TRPCs in a Chinese hamster ovary (CHO) cell model. Single CHO-S cells, expressing transfected Epo-R, were identified by detection of green fluorescent protein. Cells that express transfected TRPCs were identified by detection of blue fluorescent protein. [Ca]i was monitored with Fura Red. Epo stimulation of CHO-S cells transfected with single TRPC2 isoforms (clone 14, 17, or α) and Epo-R resulted in a significant increase in [Ca]i. This was not observed in cells transfected with Epo-R and TRPC6. In addition, coexpression of TRPC6 with TRPC2 and Epo-R inhibited the increase in [Ca]i observed after Epo stimulation. Immunoprecipitation experiments demonstrated that TRPC2 associates with TRPC6, indicating that these TRPCs can form multimeric channels. These data demonstrate that specific TRPCs are expressed in primary erythroid cells and that two of these channels, TRPC2 and TRPC6, can interact to modulate calcium influx stimulated by erythropoietin.",
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Chu, X, Tong, Q, Cheung, JY, Wozney, J, Conrad, K, Mazack, V, Zhang, W, Stahl, R, Barber, DL & Miller, B 2004, 'Interaction of TRPC2 and TRPC6 in Erythropoietin Modulation of Calcium Influx', Journal of Biological Chemistry, vol. 279, no. 11, pp. 10514-10522. https://doi.org/10.1074/jbc.M308478200

Interaction of TRPC2 and TRPC6 in Erythropoietin Modulation of Calcium Influx. / Chu, Xin; Tong, Qin; Cheung, Joseph Y.; Wozney, Jocelyn; Conrad, Kathleen; Mazack, Virginia; Zhang, Wenyi; Stahl, Richard; Barber, Dwayne L.; Miller, Barbara.

In: Journal of Biological Chemistry, Vol. 279, No. 11, 12.03.2004, p. 10514-10522.

Research output: Contribution to journalArticle

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AU - Tong, Qin

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AU - Conrad, Kathleen

AU - Mazack, Virginia

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Chu X, Tong Q, Cheung JY, Wozney J, Conrad K, Mazack V et al. Interaction of TRPC2 and TRPC6 in Erythropoietin Modulation of Calcium Influx. Journal of Biological Chemistry. 2004 Mar 12;279(11):10514-10522. https://doi.org/10.1074/jbc.M308478200