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

Abstract

Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca(2+)](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 [Ca(2+)](i), which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCgamma1 by RNA interference inhibited the Epo-stimulated increase in [Ca(2+)](i) in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCgamma, and substitution of predicted PLCgamma Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCgamma and inhibited the rise in [Ca(2+)](i). Substitution of Tyr(226) alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca(2+)](i) but not the association of PLCgamma with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP(3)). To determine whether IP(3) is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP(3) receptor (IP(3)R) binding domains. In cells expressing TRPC3 with mutant IP(3)R binding sites and Epo receptor, interaction of IP(3)R with TRPC3 was abolished, and Epo-modulated increase in [Ca(2+)](i) was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCgamma-mediated process that requires interaction of PLCgamma and IP(3)R 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.

FIGURE 1.

Endogenous expression of TRPC3 in human hematopoietic cells. Western blotting was performed on lysates from UT-7 and TF-1 Epo-responsive cell lines, from CD34⁺ cells and from day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein were loaded in each lane. A, immunoblotting with anti-TRPC3 antibody demonstrated increased expression of TRPC3 in primary human erythroid cells during erythroid differentiation. Blots were probed with anti-actin antibody to compare loading of lanes. Representative results of four experiments are shown. B, densitometry was used to quantitate TRPC3 and actin bands from four experiments of lysates from CD34⁺ cells and day 10 and 14 BFU-E-derived erythroblasts. The TRPC3/actin ratio was calculated and normalized to CD34⁺ cells to allow comparison between experiments, and the mean normalized ratio ± S.E. was determined. TRPC3 expression was significantly less in CD34⁺ cells than in day 10 erythroblasts (p < 0.02). C, immunoblotting with anti-Epo-R antibody demonstrated greatest Epo-R expression in day 10 primary erythroblasts. Representative results of three experiments are shown.

FIGURE 2.

Dose response and time course of [Ca²⁺]_i after Epo stimulation of HEK 293T cells transfected with TRPC3 and Epo-R. A, Epo dose response. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 0–40 units/ml Epo. [Ca²⁺]_i was measured at 2–5-min intervals for 20 min. The peak percentage increase of [Ca²⁺]_i above base line was calculated for each cell. Mean ± S.E. of the peak percentage increase above base line at each Epo dose is shown. 13–20 individual cells were studied at each dose in two experiments. *, a significant increase in [Ca²⁺]_i compared with cells treated for 20 min with PBS (p < 0.0001). B and C, time course. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 40 units/ml Epo. [Ca²⁺]_i was measured at 5-s intervals for the first 30 s, at 15-s intervals for the next 60 s, and then at 2-min intervals for 20 min after simulation with Epo (B) or PBS (C). Mean ± S.E. [Ca²⁺]_i (nm) of 20 (Epo) or 15 (PBS) cells measured at each time point is shown. *, a significant increase in [Ca²⁺]_i in Epo-treated cells compared with those stimulated with PBS.

FIGURE 3.

Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A, cells were treated with 40 units/ml Epo in the presence (0.68 mm) or absence (2 mm EGTA) of extracellular calcium. B, cells were treated with or without 40 units/ml Epo in the presence of 2 mm EGTA, and 3 mm CaCl₂ was added at 10 min. [Ca²⁺]_i was measured at 2–5-min intervals for 20 min, and the peak percentage increase of [Ca²⁺]_i above base line was calculated for each cell. Mean ± S.E. of the peak percentage increase in [Ca²⁺]_i at different time points is shown. 21–38 individual cells were studied for each condition in two experiments. *, a significant increase in [Ca²⁺]_i compared with cells treated for 20 min with PBS (p < 0.0001).

FIGURE 4.

U-73122 but not U-73343 inhibits the Epo-stimulated rise in [Ca²⁺]_i in primary human erythroblasts and HEK cells. A, day 10 BFU-E-derived cells. BFU-E-derived erythroblasts were removed from methyl-cellulose culture on day 10 and loaded with Fura Red. Where indicated, some cells were pretreated with the active PLC inhibitor U-73122 or the inactive inhibitor U-73343 during Fura Red loading. B, HEK 293T cells transfected with TRPC3 and Epo-R. Transfected HEK 293T cells were pretreated with the active PLC inhibitor U-73122, the inactive inhibitor U-73343, or medium during Fura Red loading. For all experimental groups, base-line [Ca²⁺]_i and the peak percentage increase in [Ca²⁺]_i above base line were calculated for each cell following monitoring at 2-min intervals for 20 min with digital video imaging. Mean ± S.E. of the peak percentage increase for each experimental condition is shown. 7–28 individual cells were studied for each condition in two experiments. **, a significant difference in [Ca²⁺]_i compared with cells treated with Epo (p < 0.0001).

FIGURE 5.

Western blot of HEK 293T cells transfected with siRNA targeted to PLCγ. Lysates were prepared from HEK 293T cells transfected (Tx'd) with or without BFP-TRPC3 and Epo-R, and siRNA was targeted to PLCγ or control siRNA. Blots were probed with anti-PLCγ, anti-TRPC3, anti-Epo-R, and anti-tubulin antibodies, followed by ECL.

FIGURE 6.

Association of TRPC3 and TRPC3-F4 with PLCγ or Epo-R. A, PLCγ and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation (IP) was performed on lysates with anti-PLCγ or anti-V5 antibodies or normal rabbit serum (NRS). Western blotting (WB) was performed after immunoprecipitation with anti-PLCγ or anti-V5 antibodies. Representative results of five experiments are shown. B, immunoprecipitation was performed on lysates from primary human erythroid cells at Phase II day 8 of liquid culture with anti-PLCγ or anti-TRPC3 antibodies or normal rabbit serum. Western blotting of eluates was performed with anti-PLCγ or anti-TRPC3 antibodies. C, Epo-R and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation was performed on lysates with anti-Epo-R or anti-V5 antibodies or normal rabbit serum. Western blotting was performed with anti-Epo-R or anti-V5 antibodies. Representative results of three experiments are shown. Tx'd, transfected.

FIGURE 7.

Interaction of TRPC3 with PLCγ SH2 binding site substitutions with PLCγ. PLCγ and V5-TRPC3, V5-TRPC3-F4, or V5-TRPC3-Y226F were expressed in HEK 293T cells. Immunoprecipitation (IP) was performed on lysates with anti-PLCγ or anti-V5 antibodies, followed by Western blotting (WB). Representative results of six experiments are shown. Tx'd, transfected.

FIGURE 8.

Schematic model of TRPC3 transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP₃R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa, amino acids.

FIGURE 9.

Association of TRPC3 IP₃R binding mutants with IP₃R. IP₃R type II and V5-TRPC3, V5-TRPC3-DEL, or V5-TRPC3-SUB were expressed in HEK 293T cells. Immunoprecipitation (IP) was performed on lysates with anti-V5 or anti-IP₃R antibodies or normal rabbit serum (NRS). Western blotting (WB) of precipitated protein was performed with anti-IP₃R or anti-V5 antibodies. Representative results of three experiments are shown.

FIGURE 10.

Plasma membrane externalization of TRPC3 detected by cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-DEL, V5-TRPC3-SUB, or V5-TRPC3-F4 and Epo-R. Lysates were prepared, and immunoprecipitation (IP) was performed with anti-V5 antibody. Western blotting (WB) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and anti-V5-HRP to detect total TRPC3. Representative results of two experiments are shown. Tx'd, transfected.