Panx3 functions as an ER Ca²⁺ channel, and activates the CaM and Akt pathways. (A, a and b) Panx3 ER Ca²⁺ channel. C2C12 cells stably transfected with pEF1 (black) or pEF1/Panx3 (red) expression vectors were analyzed for ATP-stimulated [Ca²⁺]_i in a time course (A, a). Primary calvarial cells were transiently transfected with pEF1 (black) or pEF1/Panx3 (red) expression vectors (A, b). The data shown are representative of at least four different experiments. (A, c) [Ca²⁺]_i levels during differentiation of C2C12 cells. Untransfected and stably transfected cells with pEF1/Panx3 or shPanx3 vectors were cultured with BMP2 at the indicated days. [Ca²⁺]_i levels in pEF1/Panx3-transfected cells were much higher than in C2C12 cells, whereas those in shPanx3 cells were lower. *, P < 0.05; **, P < 0.01. Error bars represent the mean ± SD, n = 3. (B and C) Panx3 activates the CaM/NFATc1 signaling pathways. C2C12 cells or primary calvarial cells were stably and transiently transfected with pEF1 and pEF1/Panx3 vectors, respectively, then incubated for 1 h with BMP2, and the levels of the signal molecules were analyzed by Western blotting. For shPanx3 inhibition experiments, stably transfected C2C12 cells or transiently transfected primary calvarial cells with sh control and shPanx3 RNA were cultured for 1 d in the presence of BMP2.