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) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (alpha beta gamma-rENaC), truncated Liddle's mutants (alpha beta(T)gamma-, alpha beta gamma(T)-, and alpha beta(T)gamma(T)-rENaC), and point mutants (alpha beta(Y)gamma-, alpha beta gamma(Y)-rENAC) expressed in Xenopus oocytes, The chord conductances of alpha beta(T)gamma-, alpha beta gamma(T)-, and alpha beta(T)gamma(T)-rENaC were 2- or 3-fold greater than for wt alpha beta gamma-rENaC. Introduction of peptides into oocytes expressing alpha beta(T)gamma-, alpha beta gamma(T)-, and alpha beta(T)gamma(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na+ conductances, with apparent dissociation constants (K-d) ranging from 1700 to 160 mu M, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt alpha beta gamma-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as 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 single channel properties of either wt or single-point mutants of rENaC, Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alpha beta(T)gamma(T)) expressed in Xenopus oocytes but not the single-point mutants (alpha beta(Y)gamma or alpha beta gamma(T)). Moreover, the blocking effect of both peptides in combination on alpha beta(T)gamma(T)-rENaC was synergistic.