%0 Journal Article
%T A Novel Mechanism in Regulating the Alpha-Subunit of the Epithelial Sodium Channel (¦Á ENaC) by the Alternatively Spliced Form ¦Á ENaC-b
%A Marlene F. Shehata
%J Biochemistry Insights
%D 2009
%I 
%X Introduction: In Dahl rats¡¯ kidney cortex, the alternatively spliced form of the epithelial sodium channel ¦Á subunit (¦Á ENaC-b) is the most abundant mRNA transcript (32+/-3 fold ¦Á ENaC-wt) as was investigated by quantitative RT-PCR analysis. ¦Á 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 ¦Á ENaC-b by testing its potential expression in COS7 cells as well as its impact on ¦Á ENaC-wt expression levels when co-expressed in COS7 cells in a dose-dependent manner.Methods: Using RT-PCR strategy, the full-length wildtype ¦Á ENaC transcript and the alternatively spliced form ¦Á 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 ¦Á ENaC-b in vitro, and to determine if an interaction between ¦Á ENaC-b and ¦Á ENaC-wt occurs in vitro, and finally to demonstrate if degradation of ¦Á ENaC-wt protein does occur.Results: ¦Á ENaC-b is translated in COS7 cells. Co-expression of ¦Á ENaC-b together with ¦Á ENaC-wt reduced ¦Á ENaC-wt levels in a dose-dependent manner. ¦Á ENaC-wt and ¦Á ENaC-b appear to form a complex that enhances the degradation of ¦Á ENaC-wt.Conclusions: Western blots suggest a novel mechanism in ¦Á ENaC regulation whereby ¦Á ENaC-b exerts a dominant negative effect on ¦Á ENaC-wt expression. This is potentially by sequestering ¦Á ENaC-wt, enhancing its proteolytic degradation, and possibly explaining the mechanism of salt-resistance in Dahl R rats.
%K ¦Á ENaC-wt
%K ¦Á ENaC-b
%K COS7 cells
%K transfection
%K expression
%K binding
%K dose-dependent response
%K salt-sensitive hypertension
%K Dahl rats
%U http://www.la-press.com/a-novel-mechanism-in-regulating-the-alpha-subunit-of-the-epithelial-so-a1416