Fig. 4.

The pUS21 forms a Ca²⁺-permeable channel. (A) Expression of pUS21 reduces the agonist releasable Ca²⁺ content. (Left) Tetracycline-inducible expression of pUS21HA in TRex-293-US21HA cells was verified by immunoblotting. Protein extracts were from NI cells or I (1 μg/mL for 48 h) cells. (Middle) Time course of intracellular Ca²⁺ signals [R (340/380) in tetracycline-induced TRex-293-US21HA cells (red) compared with NI cells (black)]. Ca²⁺ signals were measured in ionomycin/thapsigargin-stimulated Fura-2 AM-loaded cells. (Right) Box and whisker plots show the maximum cytosolic Ca²⁺ concentrations ± SD of three experiments. ***P < 0.0001, vs. TRex-293-US21HA cells NI. (B) Asp201 is important for pUS21 Ca²⁺ channel activity. (Left) Tetracycline-inducible expression of pUS21HA, pUS21HA-D178N, and pUS21HA-D201N in TRex-293-derived cell lines was verified by immunoblotting. Protein extracts were from NI cells or I cells. (Middle) Representative Ca²⁺ traces assessed in tetracycline-induced TRex-293-US21HA cells (red), TRex-293-US21HA-D178N cells (green), or TRex-293-US21HA-D201N cells (blue) compared with NI cells (black). Ca²⁺ signals were measured in ionomycin/thapsigargin-stimulated Fura-2 AM-loaded cells. Traces represent mean ± SD of three independent experiments. (Right) Box and whiskers plots show the maximum cytosolic Ca²⁺ concentrations ± SD of three experiments. ***P < 0.0001, *P < 0.05, vs. TRex-293-US21HA cells NI.