Erythropoietin modulates calcium influx through TRPC2.
Mammalian isoforms of calcium-permeable Drosophila transient receptor potential channels (TRPC) are involved in the sustained phase of calcium entry in nonexcitable 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 < or = 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.
Chu
Xin
X
Henry Hood Research Program, The Sigfried and Janet Weis Center for Research, Geisinger Clinic, 100 N. Academy Avenue, Danville, PA 17822, USA.
Cheung
Joseph Y
JY
Barber
Dwayne L
DL
Birnbaumer
Lutz
L
Rothblum
Lawrence I
LI
Conrad
Kathleen
K
Abrasonis
Virginia
V
Chan
Yiu-Mo
YM
Stahl
Richard
R
Carey
David J
DJ
Miller
Barbara A
BA
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D006224:Cricetinae; D004921:Erythropoietin / Q000494:pharmacology*; D007473:Ion Channels; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D017467:Receptors, Erythropoietin / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050053:TRPM Cation Channels; D014162:Transfection
D015220:Calcium Channels; D007473:Ion Channels; D008565:Membrane Proteins; D017467:Receptors, Erythropoietin; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D004921:Erythropoietin; D002118:Calcium