Peptide inhibition of constitutively activated epithelial Na+ channels expressed in Xenopus oocytes

The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the beta- and/or gamma-rat epithelial sodium channel (rENaC) sub units block constitutively activated ENaC was tested by examining the effec ts of these peptides on wild-type (wt) rENaC (alpha beta gamma-rENaC), trun cated Liddle's mutants (alpha beta(T)gamma-, alpha beta gamma(T)-, and alph a beta(T)gamma(T)-rENaC), and point mutants (alpha beta(Y)gamma-, alpha bet a gamma(Y)-rENAC) expressed in Xenopus oocytes, The chord conductances of a lpha beta(T)gamma-, alpha beta gamma(T)-, and alpha beta(T)gamma(T)-rENaC w ere 2- or 3-fold greater than for wt alpha beta gamma-rENaC. Introduction o f peptides into oocytes expressing alpha beta(T)gamma-, alpha beta gamma(T) -, and alpha beta(T)gamma(T)-rENaC produced a concentration-dependent inhib ition of the amiloride-sensitive Na+ conductances, with apparent dissociati on constants (K-d) ranging from 1700 to 160 mu M, depending upon whether in dividual peptides or their combination was used. Injection of peptides alon e or in combination into oocytes expressing wt alpha beta gamma-rENaC or si ngle-point mutants did not affect the amiloride-sensitive whole-cell curren ts. The single channel conductances of all the mutant ENaCs were the same a s that of wild type (alpha beta gamma-). The single channel activities (N.P -o) of the mutants were similar to 2.2-2.6-fold greater than wt alpha beta gamma-rENaC (1.08 +/- 0.24, n = 7) and were reduced to 1.09 +/- 0.17 by 100 mu M peptide mixture (n = 9). The peptides were without effect on the sing le channel properties of either wt or single-point mutants of rENaC, Our da ta demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alpha beta(T)gamma(T)) expressed in Xenopus oocytes but not the si ngle-point mutants (alpha beta(Y)gamma or alpha beta gamma(T)). Moreover, t he blocking effect of both peptides in combination on alpha beta(T)gamma(T) -rENaC was synergistic.