Erythropoietin modulates calcium influx through TRPC2

Abstract Mammalian isoforms of calcium-permeableDrosophila 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.