Abstract
BackgroundThe amiloride-sensitive Epithelial Sodium Channel (ENaC) is critical in maintaining Na+ balance, extracellular fluid volume and long term blood pressure control. ENaC is composed of three main subunits α, β, & γ. While α ENaC is critical for channel functionality, β & γ ENaC maximize channel function. To date, there are four alternatively spliced forms of the α subunit of ENaC (α ENaC-a, -b, -c, & -d) that have been published in rats, in addition to the major α ENaC transcript. While α ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length α ENaC, α ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length α ENaC.Presentation of the hypothesisα ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length α ENaC subunit by physically binding to and trapping full-length α ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length α ENaC proteolytic degradation in a dose-dependent manner.Testing the hypothesis1) To examine the mRNA and protein abundance of α ENaC-b relative to α ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2) To compare the expression (mRNA and protein) of α ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3) To examine the putative binding of α ENaC-b proteins to full-length α ENaC in vitro and to determine the impact of such binding on full-length α ENaC expression in vitro.Implications of the hypothesisOur studies will be the first to demonstrate the over-expression of salt-sensitive α ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length α ENaC. The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats. Finally, findings from the present proposal will uncover a new mechanism by which alternative splicing may control the regulation of ENaC expression/function.
Highlights
The amiloride-sensitive Epithelial Sodium Channel (ENaC) is critical in maintaining Na+ balance, extracellular fluid volume and long term blood pressure control
The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats
ENaC is primarily composed of three subunits denoted by α, β, & γ. α ENaC subunit is critical for channel functionality, while β & γ subunits serve to maximize channel functionality
Summary
The amiloride-sensitive Epithelial Sodium Channel (ENaC) constitutes the major route for transporting the sodium ion (Na+) into the cell and is critical in the maintenance of Na+ balance, extracellular fluid volume and long term blood pressure control. We will continue our QRT-PCR experiments that demonstrated an enhanced mRNA [2] and will follow by Western analysis to examine protein expression of α ENaC-b in kidneys of Dahl R compared to S rats on high versus regular salt diet. The impact of α ENaC-b on full-length α ENaC expression has been elucidated (dose-dependent coexpression experiments) in cellular models (as recently reported in our studies [6]) These co-immunoprecipitation studies along with our previously reported western analyses will demonstrate the mechanism by which α ENaC-b contributes to a suppressed overall Na+ transport related to ENaC in Dahl R rats on high Na+ (e.g., by enhanced binding of α ENaC-b to full-length α ENaC, followed by proteolytic degradation of the latter). ▪ α ENaC-b proteins trap full-length α ENaC in the endoplasmic reticulum and enhance full-length α ENaC proteolytic degradation in a dose-dependent manner and serve as dominant negatives on α ENaC expression/activity
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