title: Interaction of TRPC2 and TRPC6 in erythropoietin modulation of calcium influx.
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 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.
authors.affiliation: Henry Hood Research Program, The Sigfried and Janet Weis Center for Research, the Geisinger Clinic, Danville, Pennsylvania 17822-2616, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Chu
authors.forename: Qin
authors.initials: Q
authors.lastname: Tong
authors.forename: Joseph Y
authors.initials: JY
authors.lastname: Cheung
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Wozney
authors.forename: Kathleen
authors.initials: K
authors.lastname: Conrad
authors.forename: Virginia
authors.initials: V
authors.lastname: Mazack
authors.forename: Wenyi
authors.initials: W
authors.lastname: Zhang
authors.forename: Richard
authors.initials: R
authors.lastname: Stahl
authors.forename: Dwayne L
authors.initials: DL
authors.lastname: Barber
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Miller
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004900:Erythroblasts / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D004921:Erythropoietin / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D010659:Phenylhydrazines / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000378:metabolism; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; D050053:TRPM Cation Channels; D014162:Transfection
chemical_list: D015220:Calcium Channels; D018076:DNA, Complementary; D007473:Ion Channels; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010659:Phenylhydrazines; D020033:Protein Isoforms; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; C492146:Trpc6 protein, mouse; C030299:phenylhydrazine; D004921:Erythropoietin; D049452:Green Fluorescent Proteins; D002118:Calcium