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

Introduction: In Dahl rats' kidney cortex, the alternatively spliced form of the epithelial sodium channel a subunit (alpha ENaC-b) is the most abundant mRNA transcript (32+/-3 fold > alpha ENaC-wt) as was investigated by quantitative RT-PCR analysis. alpha 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 alpha ENaC-b by testing its potential expression in COS7 cells as well as its impact on alpha ENaC-wt expression levels when co-expressed in COS7 cells in a dose-dependent manner. Methods: Using RT-PCR strategy, the full-length wildtype alpha ENaC transcript and the alternatively spliced form alpha 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 alpha ENaC-b in vitro, and to determine if an interaction between alpha ENaC-b and alpha ENaC-wt occurs in vitro, and finally to demonstrate if degradation of alpha ENaC-wt protein does occur. Results: alpha ENaC-b is translated in COS7 cells. Co-expression of alpha ENaC-b together with alpha ENaC-wt reduced alpha ENaC-wt levels in a dose-dependent manner. alpha ENaC-wt and alpha ENaC-b appear to form a complex that enhances the degradation of alpha ENaC-wt. Conclusions: Western blots suggest a novel mechanism in alpha ENaC regulation whereby alpha ENaC-b exerts a dominant negative effect on alpha ENaC-wt expression. This is potentially by sequestering alpha ENaC-wt, enhancing its proteolytic degradation, and possibly explaining the mechanism of salt-resistance in Dahl R rats.