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Alpha-ENaC is a functional element of the hypertonicity-induced cation channel in HepG2 cells and it mediates proliferation.

ABSTRACT: The molecular correlate of hypertonicity-induced cation channels (HICCs) and their role in proliferation vs. apoptosis is a matter of debate. We report in this paper that, in whole-cell patch-clamp recordings, hypertonic stress (340-->450 mosM) reversibly increased the Na(+) conductance of HepG2 cells from 0.8 to 5.8 nS. The effect was dose-dependently inhibited by flufenamate and amiloride, known blockers of HICCs, with some 50% efficiency at 300 muM. In parallel, both drugs decreased HepG2 cell proliferation [in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and with automatic cell counting]. Small interfering RNA (siRNA) silencing of the alpha-subunit of the epithelial Na(+) channel (ENaC) reduced hypertonicity-induced Na(+) currents to 60%, whereas the rate of HepG2 cell proliferation was approximately half of that of the control. Moreover, alpha-ENaC siRNA inhibited the regulatory volume increase of HepG2 cells (measured with scanning acoustic microscopy) by 60%. In florescence-activated cell sorting measurements, silencing of alpha-ENaC led to a significant decrease in the G1 and an increase in the G2/M phase of the cell cycle, whereas the S phase was not changing. Finally (determined by a caspase 3/7 assay), HICC inhibition by flufenamate and silencing of alpha-ENaC increased the rate of apoptosis in HepG2 cells. It is concluded that alpha-ENaC is one functional element of the HICC in HepG2 cells and that the channel is an important mediator of cell proliferation; likewise, HICC blockage shifts the system from a proliferative into a rather apoptotic one. This is the first report of a role of alpha-ENaC in cell proliferation.

SUBMITTER: Bondarava M

PROVIDER: S-EPMC2704294 | biostudies-other | 2009 Aug

REPOSITORIES: biostudies-other

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Project description: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 alpha, beta, & gamma. While alpha ENaC is critical for channel functionality, beta & gamma ENaC maximize channel function. To date, there are four alternatively spliced forms of the alpha subunit of ENaC (alpha ENaC-a, -b, -c, & -d) that have been published in rats, in addition to the major alpha ENaC transcript. While alpha ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length alpha ENaC, alpha ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length alpha ENaC.Presentation of the hypothesisalpha 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 alpha ENaC subunit by physically binding to and trapping full-length alpha ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length alpha ENaC proteolytic degradation in a dose-dependent manner.Testing the hypothesis1) To examine the mRNA and protein abundance of alpha ENaC-b relative to alpha ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2) To compare the expression (mRNA and protein) of alpha ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3) To examine the putative binding of alpha ENaC-b proteins to full-length alpha ENaC in vitro and to determine the impact of such binding on full-length alpha ENaC expression in vitro.Implications of the hypothesisOur studies will be the first to demonstrate the over-expression of salt-sensitive alpha ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length alpha 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.

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Alpha-ENaC is a functional element of the hypertonicity-induced cation channel in HepG2 cells and it mediates proliferation.

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