A Novel Mechanism in Regulating the Alpha-Subunit of the Epithelial Sodium Channel (α ENaC) by the Alternatively Spliced Form α ENaC-b

Abstract

Introduction In Dahl rats’ kidney cortex, the alternatively spliced form of the epithelial sodium channel α subunit (α ENaC-b) is the most abundant mRNA transcript (32+/-3 fold > α ENaC-wt) as was investigated by quantitative RT-PCR analysis. α ENaC-b mRNA levels were significantly higher in Dahl R versus S rats, and were further augmented by high salt diet. Objectives In the present study, we described the molecular cloning and searched for a possible role of α ENaC-b by testing its potential expression in COS7 cells as well as its impact on α ENaC-wt expression levels when co-expressed in COS7 cells in a dose-dependent manner. Methods Using RT-PCR strategy, the full-length wildtype α ENaC transcript and the alternatively spliced form α ENaC-b were amplified, sequenced, cloned, subcloned into PCMV-sport6 expression vector, expressed and co-expressed into COS7 cells in a dose-dependent manner. A combination of denaturing and native western blotting techniques was employed to examine the expression of α ENaC-b in vitro, and to determine if an interaction between α ENaC-b and α ENaC-wt occurs in vitro, and finally to demonstrate if degradation of α ENaC-wt protein does occur. Results α ENaC-b is translated in COS7 cells. Co-expression of α ENaC-b together with α ENaC-wt reduced α ENaC-wt levels in a dose-dependent manner. α ENaC-wt and α ENaC-b appear to form a complex that enhances the degradation of α ENaC-wt. Conclusions Western blots suggest a novel mechanism in α ENaC regulation whereby α ENaC-b exerts a dominant negative effect on α ENaC-wt expression. This is potentially by sequestering α ENaC-wt, enhancing its proteolytic degradation, and possibly explaining the mechanism of salt-resistance in Dahl R rats.