Panx3 ER Ca²⁺ channel activation and its downstream signaling. C2C12 cells stably transfected with pEF1 or pEF1/Panx3 expression vectors were analyzed for ATP-stimulated [Ca²⁺]_i. Inhibitors were added to the cell culture for 30 min before ATP stimulation. In inhibition to endogenous IP3R3 expression, [Ca²⁺]_i was analyzed after 3 d of transfection of C2C12 cells with siRNA for IP3R3. (A) Panx3 ER Ca²⁺ channel independent of the IP3R ER Ca²⁺ channel. 2-APB (IP3R inhibitor; a) or U-73122 (IP3 synthesis inhibitor; b) completely inhibited Ca²⁺ release from the IP3R ER Ca²⁺ channel. The Panx3 ER Ca²⁺ channel was inhibited by 2-APB (a), whereas it was partially inhibited by U-73122 (b). siRNA for IP3R3 inhibited the IP3R3 ER Ca²⁺ channel, but not the Panx3 ER Ca²⁺ channel (c). Thapsigargin (SERCA ER Ca²⁺ pump inhibitor) completely inhibited Ca²⁺ release from the ER in pEF1/Panx3-transfected cells, whereas it partially inhibited it in pEF1-transfected cells (d). The data shown are representative of at least three different experiments. (B) PPADS inhibited the Panx3 ER Ca²⁺ channel but not the IP3R ER Ca²⁺ channel (a). Suramin completely inhibited the IP3R ER Ca²⁺ channel but partially inhibited the Panx3 ER Ca²⁺ channel (b). A combination of PPADS and suramin blocked both ER Ca²⁺ channels (c). Arrows indicate the time of ATP addition. The data shown are representative of at least three different experiments. (C) PPADS inhibition of CaM downstream signaling. Stably transfected C2C12 cells with pEF1 and pEF1/Panx3 vectors were incubated for 1 h with BMP2, with or without PPADS, and levels of phosphorylation of CaMKII (a) and Smad1/5 (b) phosphorylation were analyzed by Western blotting. The left panel in b indicates Smad1/5 phosphorylation levels in cells without BMP2 and PPADS. In the middle and right panels of b, cells were induced by BMP2.