TY - JOUR
T1 - Erythropoietin modulates calcium influx through TRPC2
AU - Chu, Xin
AU - Cheung, Joseph Y.
AU - Barber, Dwayne L.
AU - Birnbaumer, Lutz
AU - Rothblum, Lawrence I.
AU - Conrad, Kathleen
AU - Abrasonis, Virginia
AU - Chan, Yiu Mo
AU - Stahl, Richard
AU - Carey, David J.
AU - Miller, Barbara
PY - 2002/9/13
Y1 - 2002/9/13
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M205541200
DO - 10.1074/jbc.M205541200
M3 - Article
C2 - 12167663
AN - SCOPUS:0037072875
SN - 0021-9258
VL - 277
SP - 34375
EP - 34382
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 37
ER -