Erythropoietin Modulates Calcium Influx through TRPC2

Mammalian isoforms of calcium-permeableDrosophila 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 ≤ 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. Mammalian isoforms of calcium-permeableDrosophila 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 ≤ 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. erythropoietin transient receptor potential green fluorescent protein blue fluorescent protein fetal calf serum Chinese hamster ovary phosphate-buffered saline fluorescein isothiocyanate 4′,6-diamidino-2-phenylindole cytomegalovirus vomeronasal organ Erythropoietin (Epo)1 is a hematopoietic growth factor that regulates proliferation, differentiation, and viability of erythroid progenitors and precursors (1Damen J.E. Krystal G. Exp. Hematol. 1996; 24: 1455-1459PubMed Google Scholar, 2Wojchowski D.M. Gregory R.C. Miller C.P. Pandit A.K. Pircher T.J. Exp. Cell Res. 1999; 253: 143-156Crossref PubMed Scopus (191) Google Scholar, 3Cheung J.Y. Miller B.A. Nephron. 2001; 87: 215-222Crossref PubMed Scopus (57) Google Scholar). Regulation of intracellular calcium ([Ca]i) by erythropoietin is one of the signaling mechanisms controlling proliferation and differentiation of erythroid cells (4Miller B.A Scaduto R.C., Jr. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 5Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto R.C., Jr. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 6Mladenovic J. Kay N.E. J. Lab. Clin. Med. 1988; 112: 23-27PubMed Google Scholar, 7Misiti J. Spivak J.L. J. Clin. Invest. 1979; 64: 1573-1579Crossref PubMed Scopus (51) Google Scholar, 8Gillo B., Ma, Y.-S. Marks A.R. Blood. 1993; 81: 783-792Crossref PubMed Google Scholar, 9Hensold J.O. Dubyak G. Housman D.E. Blood. 1991; 77: 1362-1370Crossref PubMed Google Scholar, 10Levenson R. Housman D. Cantley L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5948-5952Crossref PubMed Scopus (84) Google Scholar). Evidence implicating calcium in control of erythroid growth and differentiation includes: (a) enhancement of Epo-induced murine erythroid colony growth by the ionophore A23187 and inhibition by treatment with EGTA, a nonspecific chelator of calcium (7Misiti J. Spivak J.L. J. Clin. Invest. 1979; 64: 1573-1579Crossref PubMed Scopus (51) Google Scholar); (b) demonstration that an increase in Ca2+ influx is an early and necessary step in the commitment to differentiation of murine erythroleukemia cells (8Gillo B., Ma, Y.-S. Marks A.R. Blood. 1993; 81: 783-792Crossref PubMed Google Scholar, 9Hensold J.O. Dubyak G. Housman D.E. Blood. 1991; 77: 1362-1370Crossref PubMed Google Scholar, 10Levenson R. Housman D. Cantley L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5948-5952Crossref PubMed Scopus (84) Google Scholar); and (c) the significant rise in [Ca]i stimulated by Epo observed at specific stages of human BFU-E differentiation (5Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto R.C., Jr. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar). Substantial evidence supports the conclusion that erythropoietin stimulates calcium influx in erythroid cells through voltage-independent calcium-permeable channel(s) (8Gillo B., Ma, Y.-S. Marks A.R. Blood. 1993; 81: 783-792Crossref PubMed Google Scholar, 10Levenson R. Housman D. Cantley L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5948-5952Crossref PubMed Scopus (84) Google Scholar,11Sawyer S.T. Krantz S.B. J. Biol. Chem. 1984; 259: 2769-2774Abstract Full Text PDF PubMed Google Scholar, 12Cheung J.Y. Elensky M.B. Brauneis U. Scaduto R.C., Jr. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 13Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. PubMed Google Scholar). of human erythroid cells, Epo stimulation calcium channel and J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. PubMed Google Scholar). the of erythropoietin to calcium influx and cell proliferation and viability via stimulation of receptor in cells to Epo-R, and Epo stimulates proliferation to the differentiation S.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells, Epo stimulated an increase in [Ca]i that was extracellular calcium Erythropoietin cell lines and Epo stimulated calcium influx in cells S. K. Jr. R. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). demonstrated an and of erythropoietin Biol. 2001; PubMed Scopus Google Scholar, A. 2001; Google Scholar, S. S. A. R. A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, K. J. J. 1999; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). Epo stimulated an increase in cell viability in growth cells, and [Ca]i. These were by that Epo and viability through activation of calcium channels K. J. J. 1999; PubMed Scopus Google Scholar). These a Epo a growth factor capable of proliferation and differentiation and the of the of erythropoietin of calcium in the mechanisms through which erythropoietin calcium entry and the of this cell growth and differentiation the in and the calcium-permeable channel(s) a transient receptor potential protein was of a of calcium-permeable channels in nonexcitable cells, the in G. Full Text Full Text PDF PubMed Scopus Google Scholar, Scholar). is in the is required and is via a protein to from a of isoforms which into Scholar). isoforms to the of of channels that necessary the classical or G. Full Text Full Text PDF PubMed Scopus Google channels (TRPC) were channels to the Epo-modulated calcium-permeable channels in human erythroblasts. These TRPC which in and greater than in high in the This of channels and which through intracellular G. Full Text Full Text PDF PubMed Scopus Google Scholar, Scholar). of of [Ca]i by erythropoietin at the single cell using to digital video (4Miller B.A Scaduto R.C., Jr. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 5Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto R.C., Jr. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 12Cheung J.Y. Elensky M.B. Brauneis U. Scaduto R.C., Jr. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 13Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. PubMed Google Scholar, B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). this RT-PCR to determine the expression of TRPC murine erythroid cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca]i B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). TRPC2 was the only TRPC in cells that the of TRPC in of TRPC2 were in hematopoietic cells. expression and of the longest TRPC2 clone 14 was examined. Cell and using an specific TRPC2 clone 14 demonstrated of erythropoietin to calcium influx through TRPC2 was using a digital video in which single cells that transfected Epo-R were by detection of green fluorescent protein cells that transfected TRPC were by detection of blue fluorescent protein and [Ca]i changes were simultaneously by Rhod-2 or Fura were from in and at Ba/F3 cells transfected with murine Epo-R were in with and HCD-57 cells were in with and CHO cells were in with were by with and were by the was and a single cell was D.L. Blood. 2001; PubMed Scopus Google Scholar, G. Exp. Hematol. Google Scholar). To erythroid cells K. K. J. Hematol. PubMed Scopus Google the cell was and with cells were by with the of the cell revealed that greater than of cells were erythroblasts. was from murine Epo-R, and CHO cells. was from using the were TRPC and TRPC was confirmed using the RT-PCR was at at at and were in and mTRPC2, and and and and and To RT-PCR to of the the were and and clone 14, and clone and in RT-PCR were and and was to an in the to the of clone 14 and of the was confirmed using in with clone 14 clone and G. R. L. 1996; Full Text Full Text PDF PubMed Scopus Google into in were with the HCD-57 cells were in of with cells were with in at and in in in serum with primary clone at by in the were with in to were using a were with an digital from and using and from Cell from CHO cells or transfected with clone 14 in Ba/F3 Epo-R cells, HCD-57 cells, and were from at and of was murine and were with a to a cell cells by and and was to were and an of was were at at and the was at at were in was using the to and Western with were B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Bell L. Miller B.A. Blood. PubMed Google Scholar). was were with clone 14 or was the pTracer-CMV an expression of a and a expression of was B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). TRPC2 clone 14 TRPC2 clone and G. R. L. 1996; Full Text Full Text PDF PubMed Scopus Google in were subcloned into pQBI50 pQBI50 a which expression of fused through a flexible linker to TRPC2 or CHO cells at were transfected with pTracer-CMV pQBI50 and in at was to an of with was and this was with with Successful transfection of CHO cells with Epo-R and TRPC was verified by detection of and in the cells with digital video (4Miller B.A Scaduto R.C., Jr. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 5Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto R.C., Jr. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 12Cheung J.Y. Elensky M.B. Brauneis U. Scaduto R.C., Jr. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 13Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. PubMed Google B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). expression of Epo-R and pQBI50 TRPC2 was and this was to the of transfected CHO cells to this of CHO cells and Successful transfection was confirmed by Western using cell of and transfected CHO cells B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). was from digital video was to [Ca]i (4Miller B.A Scaduto R.C., Jr. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 5Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto R.C., Jr. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 12Cheung J.Y. Elensky M.B. Brauneis U. Scaduto R.C., Jr. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 13Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. PubMed Google Scholar, B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). To changes in [Ca]i in transfected cells, were to the detection and with of the fluorescent Ca2+ Rhod-2 B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. K. J. PubMed Scopus Google Scholar, Res. PubMed Scopus Google in Fura J. Google Scholar, S.M. J. 1993; 64: Full Text PDF PubMed Scopus Google Scholar). Rhod-2 is a single Ca2+ and is to and at by at base was to changes in [Ca]i in CHO cells. CHO cells were loaded with Rhod-2 and stimulated with erythropoietin Rhod-2 was at base line and at and Epo to with and in Fura and J. Google S.M. J. 1993; 64: Full Text PDF PubMed Scopus Google a is to [Ca]i. transfected CHO cells were loaded with Fura in the of to at at was by that at to the which was at base line and a cells were to and Epo stimulation with to from the [Ca]i were in either with or calcium of erythropoietin to calcium influx in murine erythroid cells and erythroleukemia cell lines demonstrated (7Misiti J. Spivak J.L. J. Clin. Invest. 1979; 64: 1573-1579Crossref PubMed Scopus (51) Google Scholar, 8Gillo B., Ma, Y.-S. Marks A.R. Blood. 1993; 81: 783-792Crossref PubMed Google Scholar, 9Hensold J.O. Dubyak G. Housman D.E. Blood. 1991; 77: 1362-1370Crossref PubMed Google Scholar, 10Levenson R. Housman D. Cantley L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5948-5952Crossref PubMed Scopus (84) Google Scholar, S.T. Krantz S.B. J. Biol. Chem. 1984; 259: 2769-2774Abstract Full Text PDF PubMed Google Scholar, B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Here, to whether this influx through the classical the expression of to was in HCD-57 murine erythroleukemia cells and in Ba/F3 Epo-R cells, a hematopoietic cell line transfected with murine Epo-R and to to Epo with a rise in [Ca]i B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). was with that in murine and was a control, TRPC are in the RT-PCR was using from murine Ba/F3 Epo-R, and HCD-57 cells. are in TRPC2 was in Ba/F3 Epo-R cells and in HCD-57 cells. TRPC2 were observed was the that from of was confirmed by expression of classical TRPC was in hematopoietic cell of murine TRPC2 clone 14 clone G. J. PubMed Scopus Google and G. J. PubMed Scopus Google of isoforms by the RT-PCR in To determine which of isoforms are in Ba/F3 and HCD-57 cells, RT-PCR from Ba/F3 Epo-R, and HCD-57 cells using that are capable of either or of the the of the isoforms and the of is in are of RT-PCR are in and clone 14 were in hematopoietic cell of was confirmed by and clone were TRPC2 clone was observed with the clone TRPC2 clone was the the of this to a was To the expression and of the longest clone 14, an was to an to the of clone To in was using clone 14, clone and into of the was with These results are in of clone of the of the of this isoform the of a were Western was with of with to clone 14 or with to These results demonstrate the of to clone To determine whether is in the of hematopoietic cell were with HCD-57 cells using clone was to serum was a control Cell was by at through the cell were to to high results and demonstrate that clone 14 protein is at or in to the in cells. To the of to the were from CHO cells, clone CHO cells, Ba/F3 Epo-R cells, and HCD-57 cells. To the of clone 14 expression in the Epo-modulated calcium increase in primary erythroid cells, were from from the of revealed that Epo stimulates a rise in calcium in cells Western was with protein in the or protein of was observed in from clone CHO cells, Ba/F3 Epo-R cells, and HCD-57 cells was observed in CHO cells. of was demonstrated by with to murine Epo-R was observed in the in Ba/F3 Epo-R cells, HCD-57 cells, and observed from HCD-57 cells were in the of which in a Epo-R, Ba/F3 Epo-R cells were in and cells were from Epo the These the of clone 14 and Epo-R in erythroid cells. To determine whether clone 14 protein expression is to specific cell Western from murine and was at in or the expression of this a was observed at with RT-PCR results that clone 14 protein is in were observed to the at and protein or clone 14 only in is in to the of in the of murine To determine whether Epo is capable of calcium influx through mTRPC2, a in which single cells transfected with Epo-R by cells transfected with by and [Ca]i simultaneously in the cells with digital video CHO cells were Epo-R and to of the necessary required a growth and Epo-induced [Ca]i increase B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). CHO cells are with Epo-R and clone 14 CHO cells transfected with Epo-R were by CHO cells successful transfection with [Ca]i was in the cells by with Rhod-2 by was Rhod-2 or Fura of transfected in CHO cells was confirmed by Cell from CHO cells or CHO cells transfected with clone 14 or were Western was with to clone 14, and were and with to expression are in of with that in from or is a of to These results that CHO cells of TRPC2 clone 14 or TRPC6 orthologs or that the TRPC to with the hamster TRPC CHO cells were cotransfected with Epo-R and clone 14 CHO cells were cotransfected with Epo-R and empty pQBI50 or subcloned with [Ca]i in cells loaded with Rhod-2 was and at Epo are in Epo stimulation of CHO cells transfected with Epo-R and empty pQBI50 demonstrated a increase in [Ca]i above base line ± with B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Epo stimulation of CHO cells cotransfected with Epo-R and clone 14 resulted in a rise in [Ca]i above base line (372 ± 71%), which was significantly (p ≤ 0.0007) greater than that seen in cells cotransfected with or empty pQBI50 vector. increase in [Ca]i was seen CHO cells cotransfected with Epo-R and TRPC2 were stimulated with the of the Epo cells cotransfected with Epo-R and which was in murine hematopoietic cell the increase in [Ca]i in to Epo was from that observed in cells transfected with Epo-R and empty pQBI50 to Epo of CHO cells transfected with Epo-R and ± ± ± ± ± ± ± ± ± greater than by of (p ≤ ± ± ± ± ± ± cells were cotransfected with pTracer-CMV and with empty pQBI50 clone 14 subcloned into or subcloned into pQBI50. in cells was Epo and was at and Epo stimulation and the increase of ± are at at of cells greater than by of (p ≤ in a CHO cells were cotransfected with pTracer-CMV and with empty pQBI50 clone 14 subcloned into or subcloned into pQBI50. in cells was Epo and was at and Epo stimulation and the increase of ± are at at of cells To were in the of to from cells. CHO cells cotransfected with Epo-R and clone 14 or were loaded with [Ca]i and Epo stimulation are in significant increase in [Ca]i observed in cells was and to Epo of CHO cells transfected with Epo-R and in the of ± ± ± ± ± ± greater than by of (p ± ± ± ± ± ± cells were cotransfected with pTracer-CMV and with empty pQBI50 clone 14 subcloned into and subcloned into pQBI50. in cells was Epo stimulation and at Epo stimulation in the of and increase ± are at at of cells greater than by of (p in a CHO cells were cotransfected with pTracer-CMV and with empty pQBI50 clone 14 subcloned into and subcloned into pQBI50. in cells was Epo stimulation and at Epo stimulation in the of and increase ± are at at of cells To that the [Ca]i increase in to erythropoietin in transfected CHO cells from calcium influx than CHO cells transfected with Epo-R and TRPC2 clone 14 were stimulated by Epo in the of calcium or [Ca]i was in Fura cells in [Ca]i of cells was observed in the of extracellular calcium. calcium was at was a and significant increase (p ≤ in [Ca]i in cells with erythropoietin at of calcium to transfected CHO cells with erythropoietin increase [Ca]i. These results that erythropoietin the calcium influx through TRPC2, which that extracellular calcium was [Ca]i to Ca2+ from in to [Ca]i was at the Epo increase in [Ca]i was this CHO cells were transfected with Epo-R and with specific TRPC channels to erythropoietin calcium channel This is the of calcium signaling in which successful transfection of receptor and calcium channel were at the single cell by from and and [Ca]i modulation was with a Rhod-2 or Fura Red. This to of the is that TRPC2 and protein are hematopoietic cells. Using RT-PCR, Western blotting, and immunolocalization, demonstrated that a classical channel, mTRPC2, is in murine hematopoietic cells. TRPC2 clone 14 G. D. L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google clone G. D. L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google G. J. PubMed Scopus Google and G. J. PubMed Scopus Google Scholar). and G. D. L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, G. J. 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A. 1999; PubMed Scopus Google TRPC2 to the of receptor Erythropoietin to voltage-independent calcium channel(s) (8Gillo B., Ma, Y.-S. Marks A.R. Blood. 1993; 81: 783-792Crossref PubMed Google Scholar, 12Cheung J.Y. Elensky M.B. Brauneis U. Scaduto R.C., Jr. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 13Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. PubMed Google Scholar, B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is that Epo is capable of modulating calcium influx through TRPC2, and Epo-R are required a calcium rise in CHO cell B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google with that seen in human cells stimulated with the of this Epo stimulates an increase in calcium in Ba/F3 Epo-R cells B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and in in which TRPC2 and Epo-R are the demonstrate that TRPC2 is a the [Ca]i increase (4Miller B.A Scaduto R.C., Jr. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 5Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto R.C., Jr. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 6Mladenovic J. Kay N.E. J. Lab. Clin. Med. 1988; 112: 23-27PubMed Google Scholar, 7Misiti J. Spivak J.L. J. Clin. Invest. 1979; 64: 1573-1579Crossref PubMed Scopus (51) Google Scholar, 8Gillo B., Ma, Y.-S. Marks A.R. Blood. 1993; 81: 783-792Crossref PubMed Google Scholar, 9Hensold J.O. Dubyak G. Housman D.E. Blood. 1991; 77: 1362-1370Crossref PubMed Google Scholar, 10Levenson R. Housman D. Cantley L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5948-5952Crossref PubMed Scopus (84) Google Scholar, S.T. Krantz S.B. J. Biol. Chem. 1984; 259: 2769-2774Abstract Full Text PDF PubMed Google Scholar). CHO cells, were to expression of TRPC2 or TRPC6 with either RT-PCR or Western blotting, that CHO cells a to the Epo-R and TRPC the increase in [Ca]i observed in CHO cells, with that observed in cells transfected with TRPC6 or is of the of the Epo-induced [Ca]i increase in human (5Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto R.C., Jr. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 12Cheung J.Y. Elensky M.B. Brauneis U. Scaduto R.C., Jr. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar). CHO cells transfected with Epo-R only B.A. D.L. Bell L.L. Zhang Cheung J.Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google the of increase in [Ca]i was that either TRPC2 in CHO cells is in by RT-PCR or that channels in CHO cells and erythroid cells are TRPC2 to by calcium G. D. L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, L. Cell Biol. 2001; PubMed Scopus Google and activation by the receptor G. D. L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google and in by the in the extracellular L. Cell Biol. 2001; PubMed Scopus Google Scholar). TRPC2 to a in receptor activation in the L. G. PubMed Scopus Google through a to calcium calcium Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). the mechanisms by which Epo regulates TRPC2 were in this in erythroid cells, were to demonstrate an increase in [Ca]i in the Epo activation of TRPC by of calcium in cells. channels to by growth (a) a of by growth through of Zhang L. Cell Biol. 1999; PubMed Scopus Google and (b) by growth factor through activation of the receptor and 1999; 24: Full Text Full Text PDF PubMed Scopus Google Scholar). expression of clone 14 and Epo-R in erythroid cells. 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