Interaction of TRPC2 and TRPC6 in Erythropoietin Modulation of Calcium Influx

Autor: Jocelyn Wozney, Joseph Y. Cheung, Xin Chu, Barbara A. Miller, Richard C. Stahl, Qin Tong, Virginia Mazack, Wenyi Zhang, Kathleen Conrad, Dwayne L. Barber
Rok vydání: 2004
Předmět:
Erythrocytes
Erythroblasts
Biochemistry
Ion Channels
Mice
Transient receptor potential channel
Cricetinae
hemic and lymphatic diseases
Protein Isoforms
Cells
Cultured
TRPC
Microscopy
Confocal
Microscopy
Video
Voltage-dependent calcium channel
Reverse Transcriptase Polymerase Chain Reaction
Chinese hamster ovary cell
Brain
Transfection
Phenylhydrazines
Cell biology
TRPC Cation Channels
Cell Division
Protein Binding
medicine.drug
DNA
Complementary
Blotting
Western
Green Fluorescent Proteins
TRPM Cation Channels
chemistry.chemical_element
CHO Cells
Calcium
Biology
Cell Line
TRPC6 Cation Channel
medicine
Animals
Erythropoietin
Molecular Biology
Dose-Response Relationship
Drug
Cell Membrane
Membrane Proteins
Cell Biology
Precipitin Tests
Molecular biology
Protein Structure
Tertiary
Mice
Inbred C57BL
Alternative Splicing
Luminescent Proteins
Microscopy
Fluorescence
chemistry
Calcium Channels
Spleen
Zdroj: Journal of Biological Chemistry. 279:10514-10522
ISSN: 0021-9258
DOI: 10.1074/jbc.m308478200
Popis: 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 alpha) 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 alpha) 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.
Databáze: OpenAIRE
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