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J. Biol. Chem., Vol. 279, Issue 11, 10514-10522, March 12, 2004

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Interaction of TRPC2 and TRPC6 in Erythropoietin Modulation of Calcium Influx^*

Xin Chu, Qin Tong, Joseph Y. Cheung, Jocelyn Wozney, Kathleen Conrad, Virginia Mazack, Wenyi Zhang, Richard Stahl, Dwayne L. Barber¶, 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 and the ^¶Division of Cellular and Molecular Biology, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada

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 ) 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 ) 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.

Received for publication, August 1, 2003 , and in revised form, December 16, 2003.

^* This work was supported by the National Institutes of Health Grants DK 46778 (to B. A. M.) and HL 58672 (to J. Y. C.), grants from the Geisinger Foundation, and grants from the Canadian Institute for Health Research (to D. L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: The Henry Hood Research Program, The Sigfried and Janet Weis Center for Research, Geisinger Clinic, 100 N. Academy Dr., Danville, PA 17822-2616. Tel.: 570-271-6675; Fax: 570-271-6701; E-mail: bamiller1{at}geisinger.edu.

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