Fig. 2.

B31 growth inhibition in high K⁺ media represents KCNK channel activities on cell surface. (A) Alignment of C-terminal sequences from KCNK3 and KCNK9 channels. (B) Association of COPI and 14-3-3 proteins with KCNK3 (upper panels) and KCNK9 (lower panels). HEK293 cells transfected with HA-KCNK3 or Myc-KCNK9 were lysed and immunoprecipitated with HA or Myc Abs. The eluants were resolved by SDS–PAGE and immunoblotted for the associating β-COP and 14-3-3 as well as HA (KCNK3) or Myc (KCNK9). (C) Surface expression of KCNK3 and KCNK9. HEK293 cells were transfected with HA-tagged rat KCNK3 or Myc-tagged human KCNK9 and analyzed for cell surface expression by FCM. For KCNK3 (upper panels), the expression is shown in histograms and the Median values were determined for the total cell populations as described for Fig. 1. The histograms of KCNK3-transfected cells (filled) were overlaid with that of vector-transfected cells (unfilled). For KCNK9 (lower panels), the expression is shown in density plots (see Section 2) where x-axis indicates the fluorescence intensity in a logarithmic scale and y-axis indicates the side scatter (SSC) of the cell. The Median values were determined for the cells that were positive for the KCNK9 signal (shown in squares within the density plots). The bar graphs indicate the Median values in average ± s.d. of triplicate samples from the representative of three different experiments. (D) Growth assay of KCNK-expressing B31. The KCNK- or pYES2met vector-transformed B31 cells were plated on YNB media (pH 7.0 for KCNK3 and pH 6.50 for KCNK9) with indicated concentrations of KCl and cultured at 30 °C. The images were photographed at Day 7. (E) The effect of pH on the growth of KCNK9-expressing B31. The B31 cells transformed with vector or KCNK9 Wt were grown in the liquid YNB media with indicated pH and 400 mM KCl. (F) The effect of zinc on the growth of KCNK9-expressing B31.The B31 cells transformed with vector or KCNK9 Wt were grown in the liquid YNB media with indicated concentrations of ZnCl₂ and 400 mM KCl at pH 6.50. In both of the pH and zinc tests, the OD at 600 nm was measured at the start of and after 15 h of culture. The values after the culture was subtracted with those at the start and shown in average ± s.d. of triplicate samples from the representative of three experiments.