Fig. 3.

Myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1) mutations alter epithelial sodium channel (ENaC) activity. Distal convoluted tubule cell line mDCT-15 cells were transfected, treated with 1 μM aldosterone, and cultured on 24-mm permeable supports. The cell-attached patch configuration was used for all single-channel experiments 48 h after transfection of different mutants (see Fig. 2). A shows the representative recordings of each group. Unsurprisingly, S3A-transfected cells had the highest open probability, whereas S3D had the lowest. All differences in open probability between variants except the 1 between wild type (WT) and GA are statistically significant. In B is the open probability for all variants of MLP-transfected mDCT-15 cells with individual values and means ± SE. Numbers in parentheses represent the number of experiments. C shows that the expression levels of ENaC measured as the number of channels per patch are unchanged regardless of the construct. Channels per patch are discrete numbers, so bar graphs are more appropriate than dot plots. D shows expression of MLP-1 constructs (S3A, S3D, WT, and GA) in the 1st panel. The 2nd through 4th panels are Western blots of α-, β-, and γ-ENaC that show that expression of each of the ENaC subunits is unchanged regardless of the MLP-1 construct (representative of 4 similar blots). Molecular mass is in kilodaltons. Ab, antibody; -Vp, negative of the patch pipette potential. “c” and “o” to the left of single channel records indicate ENaC closed and open levels.