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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.

Project description:Background: Protein aggregates are degraded via the autophagy-lysosome pathway and alterations in the lysosomal system leading to the accumulation of pathogenic proteins, including aggregates of α-synuclein in Parkinson's disease (PD). The importance of the endolysosomal transient receptor potential cation channel, mucolipin subfamily 1 (TRPML1) for the lysosomal function is highlighted by the fact that TRPML1 mutations cause the lysosomal storage disease mucolipidosis type IV. In this study, we investigated the mechanism by which activation of TRPML1 affects the degradation of α-synuclein. Methods: As a model of α-synuclein pathology, we expressed the pathogenic A53Tα-synuclein mutant in HEK293T cells. These cells were treated with the synthetic TRPML1 agonist ML-SA1. The amount of α-synuclein protein was determined by immunoblots. The abundance of aggregates and autolysosomal vesicles was determined by fluorescence microscopy and immunocytochemistry. Findings were confirmed by life-cell imaging and by application of ML-SA1 and the TRPML1 antagonist ML-SI3 to human dopaminergic neurons and human stem cell-derived neurons. Results: ML-SA1 reduced the percentage of HEK293T cells with α-synuclein aggregates and the amount of α-synuclein protein. The effect of ML-SA1 was blocked by pharmacological and genetic inhibition of autophagy. Consistent with TRPML function, it required the membrane lipid PI(3,5)P2, and cytosolic calcium. ML-SA1 shifted the composition of autophagosomes towards a higher fraction of mature autolysosomes, also in presence of α-synuclein. In neurons, inhibition of TRPML1 by its antagonist ML-SI3 blocked autophagosomal clearance, whereas the agonist ML-SA1 shifted the composition of a-synuclein particles towards a higher fraction of acidified particles. ML-SA1 was able to override the effect of Bafilomycin A1, which blocks the fusion of the autophagosome and lysosome and its acidification. Conclusion: These findings suggest, that activating TRPML1 with ML-SA1 facilitates clearance of α-synuclein aggregates primarily by affecting the late steps of the autophagy, i.e., by promoting autophagosome maturation. In agreement with recent work by others, our findings indicate that TRPML1 might constitute a plausible therapeutic target for PD, that warrants further validation in rodent models of α-synuclein pathology.

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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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