Effect of replacement of γ-ENaC R138 (P1) by alanine, lysine, or glutamine on I_Na and fragments stimulated by CAP2. (A) CAP2 effect on I_Na of WT and α-, β-, and γ-R138A or α-, β-, and γ-R138K or α-, β-, and γ-R138Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 18). Results are expressed as the means ± SE. * and **, P < 0.0001. (B) Western blots of total whole cell extracts. CAP2-induced fragment pattern of WT and γ-ENaC mutant channels (top) and actin as loading control (bottom). Lane 1, uninjected eggs; lane 2, WT ENaC plus CAP2; lane 3, α-, β-, and γ-R138A ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC plus CAP2; lane 5, α-, β-, and γ-R138Q ENaC plus CAP2. A representative experiment is shown (n = 3). (C) Western blots of surface biotinylated (top), total γ-ENaC pools (bottom), and actin. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC alone; lane 5, α-, β-, and γ-R138K ENaC plus CAP2. A representative experiment is shown (n = 3).