Figure 1.

Sec24D regulates ENaC cleavage in MDCK αβγ ENaC and CFBE41o⁻ epithelial cells. MDCK αβγ-ENaC cells (A, C, E, G, and H) or CFBE41o⁻ epithelial cells (B, D, and F) were transiently transfected with nontargeted (control) or Sec24D siRNA and grown as polarized monolayers. Cells were mounted in Ussing chambers for I_sc measurements (A–F) or underwent surface biotinylation (G and H) as described under “Experimental procedures.” A and B, representative immunoblot analysis performed after completion of the I_sc measurements confirming siRNA-mediated depletion of Sec24D in MDCK αβγ-ENaC cells (A) or CFBE41o⁻ epithelial cells (B). Shown are representative I_sc traces from experiments in MDCK αβγ-ENaC (C) or CFBE41o⁻ (D) cells, with annotations depicting experimental protocol. E and F, summary of amiloride-sensitive I_sc measurements (relative to control baseline) are presented as mean ± S.D. (error bars); individual data points are depicted in gray, whereas representative traces from C and D are shown in black in E and F, respectively. Baseline I_sc represents ENaC that is at the membrane in a cleaved/active form. E, application of trypsin to the apical surface in MDCK αβγ-ENaC cells acutely activates uncleaved/nearly silent ENaC (n = 13, p = 0.0122 for baseline, p = 0.0088 for trypsin, p = ns for total). Boldface points in E denote the representative I_sc traces from C. F, I_sc measurements of Sec24D-depleted CFBE41o⁻ epithelial cells demonstrating decreased baseline ENaC-mediated I_sc (n = 19, p = 0.0026). Boldface points in F denote the representative I_sc traces from D. G, representative experiment demonstrating unchanged apical surface expression of β-ENaC by surface biotinylation. F, densitometric quantification of β-ENaC surface expression relative to control (n = 5 independent experiments, p = ns by Wilcoxon signed-rank test).