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Originally published In Press as doi:10.1074/jbc.M205541200 on July 11, 2002
J. Biol. Chem., Vol. 277, Issue 37, 34375-34382, September 13, 2002
Erythropoietin Modulates Calcium Influx through TRPC2*
Xin
Chu ,
Joseph Y.
Cheung §,
Dwayne L.
Barber¶ ,
Lutz
Birnbaumer**,
Lawrence I.
Rothblum ,
Kathleen
Conrad ,
Virginia
Abrasonis ,
Yiu-mo
Chan ,
Richard
Stahl ,
David J.
Carey , and
Barbara A.
Miller  §§
From The Henry Hood Research Program, The Sigfried
and Janet Weis Center for Research, and the Departments of
§ Medicine and  Pediatrics, the
Geisinger Clinic, Danville, Pennsylvania 17822, the ¶ Division of
Cellular and Molecular Biology, Ontario Cancer Institute, Toronto,
Ontario M5G 2M9, Canada, and the ** Division of Intramural
Research, NIEHS, National Institutes of Health,
Research Triangle Park, North Carolina 27709
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 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.
*
This work was supported by National Institutes of Health
Grants DK 46778 (to B. A. M), HL 58672 (to J. Y. C.), GM 46991 (to L. I. R.), and NS 21925, NS 37716, and NS 41363 (to D. J. C.) and
grants from the Geisinger Foundation (to B. A. M., J. Y. C., L. I. R., Y. C., and D. J. C.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Research Scientist of the National Cancer Institute of Canada.
§§
To whom correspondence should be addressed: The Henry Hood
Research Program, The Sigfried and Janet Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822-2616. Tel.:
570-271-6675; Fax: 570-271-6701; E-Mail:
bamiller1{at}geisinger.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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