Effect of the point mutation within the consensus K⁺ filter sequence on TASK3 currents expressed in mouse embryonic fibroblast cells. (A) Cell surface expression of the wild-type and mutant TASK3 by indirect immunofluorescence. (B) K⁺ channel activity of the stable cell lines expressing wild-type or mutant TASK3 individually or together measured by FLIPR assay. Twenty thousand cells were plated in a 384-well plate and grown for 24 h. Cells were loaded with the voltage-sensitive fluorescence dye for 30 min and assayed on FLIPR in assay buffer containing 90 mM [K⁺]. (C) TASK3 whole cell currents were recorded from the stable cell line expressing either wild-type or mutant TASK3. Pipette and bath solutions contained 140 and 5 mM K⁺, respectively. Membrane potential was held at -60 mV and stepped from -80 to +60 mV in 10-mV increments.

Effect of the point mutation within the consensus K⁺ filter sequence on K⁺ current of TASK3 expressed in Xenopus oocyte. (A) TASK3^G95E does not exhibit any measurable K⁺ current. (B) Expression of TASK3^G95E inhibits K⁺ current of the wild-type TASK3. Shown are whole cell currents in response to voltage ramp between -120 and +40 mV recorded from Xenopus oocytes 48 h after injection with individual wild-type or mutant TASK3 cRNA or in combination as indicated. (Left) Representative traces of whole cell currents recorded in response to 175-ms voltage ramp between -120 and +40 mV. (Right) Average currents (±SE) of all of the oocytes tested in each group. P value was determined by a Student's t test. The extracellular [K⁺] concentration was 40 mM (A) and 2 mM (B).

The inactivating mutation of TASK3 abolished its oncogenic functions. (A) Cell proliferation in low serum. Cell growth rate is expressed as absorbance at 490 nm. Data are representative of two independent experiments (n = 8 for each experiment). (B) Apoptosis assay (DNA fragmentation ELISA). Apoptosis was induced by treatment of cells with 1 ng/ml TNF for 16 h. Cell apoptosis is expressed as enhancement factor (fold over untreated cells in each group). Data are representative of three independent experiments (n = 3 for each experiment). (C) Tumorigenicity in nude mice. Mouse embryonic fibroblast cells stably transfected with vector alone, wild-type, or mutant TASK3 individually or together were injected (10⁶ cells per injection) into nude mice (two experiments, n = 10 for each cell line). Tumor formation was monitored. The tumors were measured each week at their longest points by using a caliper. The bar in the graph represents the average tumor size for each group. (D)K⁺ channel activity in excised tumors. Tumors were excised and cultured for a week, and the TASK3 channel activity was measured by FLIPR assay as describe in the legend for Fig. 2. (E) Ben cell proliferation. Five thousand Ben or BenTASK3^G95E cells were plated in a 96-well plate, and the cell proliferation rated was determined as in A.