TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells

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 PLC gamma 1 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 gamma, and substitution of predicted PLC gamma Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLC gamma and inhibited the rise in [Ca2+](i). Substitution of Tyr(226) alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca2+](i) but not the association of PLC gamma 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 gamma-mediated process that requires interaction of PLC gamma and IP3R with TRPC3. They also show that TRPC3 Tyr(226) is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.