Project

ion channels; surface expression; ubiquitin; ERAD; sodium homeostasis; hypertension; deubiquitylation; multimeric membrane protein; sodium transport; kidney; ER-associated degradation; quality control

Lead
Lay summary
In most developed countries, excess salt intake is a major risk factor of hypertension, one of the most common diseases in the human population, affecting over 1 billion people worldwide.The kidney is able to precisely regulate the balance between uptake and excretion of sodium into and from the blood by regulating sodium transport in the kidney. This transport involves the regulated action of a sodium channel (called ENaC), which forms a pore across membranes and allows passage of sodium. Because sodium is the most important cation in the blood, changes in its concentration have direct consequences on blood volume and hence on the arterial blood pressure. The sodium channel is regulated by hormonal mechanisms, and can be strongly disturbed in diseases such as Liddle's syndrome, an inherited form of human hypertension We have largely contributed to the understanding of this regulation, by showing that the activity of this sodium channel is controlled by a mechanism called ubiquitylation, in which a small protein (named ubiquitin) is linked to the ENaC channel, promoting its internalization and degradation. Interestingly, ubiquitylation is reversible, and it is the balance between ubiquitylation and deubiquitylation that controls the channel's activity.Aim:In this project we want to study in detail the mechanisms of deubiquitylation both in vitro and in vivo, in epithelial cells derived from the kidney, and in mouse models, in which ENaC deubiquitylation has been inactivated. Moreover, we will investigate the role of ubiquitylation in the quality control of proper ENaC synthesis and surface expression.Significance:The process addresses fundamental issues with respect to the regulation of sodium balance (and its consequences for maintenance of blood pressure), and it treats novel biological issues with respect to the ubiquitylation/deubiquitylation and quality control of ion channels.

Direct link to Lay Summary

Last update: 21.02.2013

Number	Title	Start	Funding scheme

Excess salt intake is a major risk factors of hypertension, one of the most common diseases in the human population, affecting over 1 billion individuals worldwide. The aldosterone-sensitive distal nephron (ASDN) is able to finely tune the Na(+ ) balance in extracellular fluids (including the blood) by regulating transepithelial Na(+) transport. In this context the epithelial Na(+) channel (ENaC) is highly regulated by a number of hormonal pathways, including aldosterone, vasopressin, and IGF/insulin. We have significantly contributed to the understanding of these regulatory pathways by demonstrating that ubiquitylation plays a central role in ENaC regulation. We showed that the ubiquitin-protein ligase Nedd4-2, interacts with and ubiquitylates ENaC, leading to channel internalization. We then demonstrated that this mechanism is impaired in Liddle’s syndrome, an inherited form of human hypertension, in which mutations in the genes encoding the ENaC subunits inactivate the binding sites for Nedd4-2, and consequently cause accumulation of channels at the cell surface. We have also provided evidence that ENaC ubiquitylation is regulated by several, potentially synergistic pathways: 1) aldosterone-induced Sgk1 kinase phosphorylates Nedd4-2, thereby promoting 14-3-3 binding and interference with Nedd4-2/ENaC binding, 2) dietary Na+ dependant variation of Nedd4-2 expression in the ASDN, and 3) aldosterone-induced expression of Usp2-45, a deubiquitylating enzyme able to deubiquitylate ENaC, and to promote increased cell surface expression as well as proteolytic activation of ENaC. Recently, a Nedd4-2 KO mouse generated by our collaborators proofed the concept that Nedd4-2 plays a role in salt-dependant hypertension. However, other questions remain pertinent such as the in vivo contribution of each Sgk1 and/or Usp2-45 in ENaC regulation, or the involved molecular mechanisms which we just begin to understand. Another, so far neglected mechanism in ENaC regulation involves the assembly, quality control and ER associated degradation (ERAD), which we believe to play another important role to control ENaC targeting to the cell surface. Hypothesis:ENaC is regulated by 2 different mechanisms of ubiquitylation: one involving regulated Nedd4-2 dependant multi-mono-ubiquitylation, and the other one implying ERAD dependant polyubiquitylation, important for channel export to the plasma membrane.Specific aims:1) To establish the molecular mechanism and the role of Usp2-45 in transepithelial Na+ transport both in cells derived from the CCD and in vivo models and to study the relation between Sgk1 and Usp2-45 in Na+ homeostasis in vivo in novel mice models.Methods: Gene expression/suppression in cell lines. Investigation of renal parameters of KO mice2) To characterize the molecular machinery that is involved in ER associated retention and degradation of the epithelial Na+ channel ENaC and its possible role in ENaC regulation.Methods: Identification and characterization of proteins involved in ER associated retention and degradation of ENaC. Use of various cell lines (including renal epithelial cells) for functional and biochemical analysis.Expected value of project:The project addresses fundamental issues with respect to the physiological/pathophysiological regulation of renal Na(+) homeostasis (and its consequences for maintenance of blood pressure), and it treats novel cell biological issues with respect to ubiquitylation/deubiquitylation and ERAD in the control of ion channels.