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J. Biol. Chem., Vol. 283, Issue 16, 10385-10395, April 18, 2008

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TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells^*

Qin Tong, Iwona Hirschler-Laszkiewicz, Wenyi Zhang, Kathleen Conrad, David W. Neagley, Dwayne L. Barber, Joseph Y. Cheung^¶, and Barbara A. Miller^||1

From the Departments of Pediatrics and ^||Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, the Division of Stem Cells and Developmental Biology, Ontario Cancer Institute, Ontario M5G 2M9, Canada, and the ^¶Division of Nephrology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca²⁺]_i) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca²⁺]_i, which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLC1 by RNA interference inhibited the Epo-stimulated increase in [Ca²⁺]_i in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLC, and substitution of predicted PLC Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLC and inhibited the rise in [Ca²⁺]_i. Substitution of Tyr²²⁶ alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca²⁺]_i but not the association of PLC with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP₃). To determine whether IP₃ is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP₃ receptor (IP₃R) binding domains. In cells expressing TRPC3 with mutant IP₃R binding sites and Epo receptor, interaction of IP₃R with TRPC3 was abolished, and Epo-modulated increase in [Ca²⁺]_i was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLC-mediated process that requires interaction of PLC and IP₃R with TRPC3. They also show that TRPC3 Tyr²²⁶ is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.

Received for publication, December 14, 2007 , and in revised form, February 6, 2008.

^* This work was supported by National Institutes of Health Grants R01 DK46778 (to B. A. M.), R01 HL58672 (to J. Y. C.), and R01 HL74854 (to J. Y. C.) and by the Four Diamonds Fund of the Pennsylvania State University College of Medicine and 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.

¹ To whom correspondence should be addressed: Dept. of Pediatrics, Milton S. Hershey Medical Center, P.O. Box 850, Hershey, PA. Tel.: 717-531-4654; Fax: 717-531-4789; E-mail: bmiller3{at}psu.edu.

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This article has been cited by other articles:

				I. Hirschler-Laszkiewicz, Q. Tong, K. Conrad, W. Zhang, W. W. Flint, A. J. Barber, D. L. Barber, J. Y. Cheung, and B. A. Miller TRPC3 Activation by Erythropoietin Is Modulated by TRPC6 J. Biol. Chem., February 13, 2009; 284(7): 4567 - 4581. [Abstract] [Full Text] [PDF]

				J. Abramowitz and L. Birnbaumer Physiology and pathophysiology of canonical transient receptor potential channels FASEB J, February 1, 2009; 23(2): 297 - 328. [Abstract] [Full Text] [PDF]