Fig. 1.

B31 tolerance to high K⁺ media represents the loss of Kir2.1 activity on cell surface. (A) Surface expression of Kir2.1 channels. HEK293 cells were transiently transfected with HA-tagged Kir2.1 constructs or pCDNA3.1(+) vector alone. Cells stained with the HA Ab followed by Alexa Fluor 488-conjugated secondary Ab were analyzed by flow cytometry (FCM). Histograms from the representative samples are shown (left panels). The x-axis indicates the fluorescence intensity in a logarithmic scale and y-axis indicates the cell number. The histograms of Kir2.1-transfected cells (filled) were overlaid with that of vector-transfected cells (unfilled). The bar graph (right panel) shows the Median values for the total cell populations determined by FlowJo software to compare the relative surface intensity of Wt and mutant Kir2.1 channels. The values indicate average ± s.d. of triplicate samples from the representative of three experiments. The lower panel shows the total expression levels of Kir2.1 proteins. Total lysates from HEK293 cells transfected with HA-Kir2.1 constructs were resolved by SDS–PAGE and immunoblotted for HA. (B) Growth assay of Kir2.1-expressing B31. The Kir2.1- or pYES2met vector-transformed B31 cells were plated on YNB media (pH 6.50) with the indicated concentrations of KCl and cultured at 30 °C. The images were photographed at Day 5. (C) Expression of Kir2.1 channels in B31 cells. The proteins were extracted from B31 cells transformed with pYES2met vector or the indicated Kir2.1 constructs. The samples were resolved by SDS–PAGE and immunoblotted for Kir2.1 (upper panel). The lower panel shows the Ponceau S staining of the transfer membrane, indicating similar loading of the proteins for each sample.