Effect of α-, β-, and γ-R138A-ENaC mutant on I_Na and fragments stimulated by CAP2. (A) (Top) Surface biotinylated γ-ENaC CT 75-kD fragment induced by CAP2 was visualized by Western blot analysis using an anti-V5 monoclonal antibody. (Bottom) Total whole cell lysates (input 4%) were probed for actin as loading control. FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138A ENaC alone; lane 5, α-, β-, and γ-R138A ENaC plus CAP2. A representative experiment is shown (n = 3). (B) CAP2-mediated I_Na of WT and α-, β-, and γ-R138A mutant channels were measured as described in Results. Batches of oocytes were extracted from six different frogs (n = 36). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Semi-quantification of the 75-kD γ-ENaC surface fragment induced by CAP2 (IOD, integrated optic density; n = 4).