The amiloride-sensitive epithelial sodium channel (ENaC) plays a central role in the maintenance of sodium homeostasis, it constitutes the main pathway for reabsorption of sodium in tight epithelia such as the distal nephron. The importance of the sodium channel in the maintenance of extracellular volume and blood pressure is underscored by the finding of mutations in the human genes that either active channels leading to hypertension (Liddle's syndrome), or conversely, decrease activity of channels leading to salt-wasting and hypovolemic states (pseudohypoaldosteronism). Elucidation of the mechanisms that normally regulate the function and expression of ENaC is of major importance for the understanding of blood volume maintenance in physiological and pathological conditions. The long term objectives of this work are to understand the molecular mechanisms of channel function and regulation. The specific goals of this proposal are: 1) to identify functional domains using the differences in properties exhibited by channels formed by ab and ag subunits. Mapping of functional domains will be performed using chimeras generated between the b and g subunits; 2) to elucidate the mechanics by which aldosterone mediates phosphorylation of ENaC. Aldosterone increase sodium permeability by increasing the abundance of sodium channels and by activating pre-existing channels. However, the mechanism(s) that mediate the activation of channels is still unknown. We propose that aldosterone induced phosphorylation is one of the mechanisms that activates pre-existing channels; 3) to understand the regulation of expression of ENaC by ubiquitination. We will examine the hypothesis that ubiquitination participates in endocytosis at the plasma membrane and in degradation of channels in intracellular compartments. Injected Xenopus oocytes and transfected cells will provide the expression systems for wild-type and mutant channels in which we will examine the functional properties, levels of expression, cellular distribution and rates of biosynthesis and degradation of channels. Experiments are designed to examine the activity and properties of channels by electrophysiologycal techniques. Ensembles of channels will be studied using the two micro-electrode voltage clamp and single channels using the patch-clamp technique. Biochemical, immunological, and molecular biological approaches will be applied to examine the state of phosphorylation and ubiquitination of the channel. These studies will identify important functional domains in the sodium channel, and are the first to explore two novel regulatory mechanisms of the function and expression of sodium channels.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054062-04
Application #
6381158
Study Section
General Medicine B Study Section (GMB)
Program Officer
Scherbenske, M James
Project Start
1998-06-01
Project End
2003-05-31
Budget Start
2001-06-01
Budget End
2002-05-31
Support Year
4
Fiscal Year
2001
Total Cost
$275,368
Indirect Cost
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Li, Tianbo; Yang, Youshan; Canessa, Cecilia M (2010) Two residues in the extracellular domain convert a nonfunctional ASIC1 into a proton-activated channel. Am J Physiol Cell Physiol 299:C66-73
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Arteaga, Maria Francisca; Wang, Lin; Ravid, Tommer et al. (2006) An amphipathic helix targets serum and glucocorticoid-induced kinase 1 to the endoplasmic reticulum-associated ubiquitin-conjugation machinery. Proc Natl Acad Sci U S A 103:11178-83
Coric, Tatjana; Hernandez, Nelmary; Alvarez de la Rosa, Diego et al. (2004) Expression of ENaC and serum- and glucocorticoid-induced kinase 1 in the rat intestinal epithelium. Am J Physiol Gastrointest Liver Physiol 286:G663-70
Alvarez de la Rosa, Diego; Canessa, Cecilia M (2003) Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells. Am J Physiol Cell Physiol 284:C404-14
Alvarez de la Rosa, D; Coric, T; Todorovic, N et al. (2003) Distribution and regulation of expression of serum- and glucocorticoid-induced kinase-1 in the rat kidney. J Physiol 551:455-66
Alvarez de la Rosa, Diego; Li, Hui; Canessa, Cecilia M (2002) Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells. J Gen Physiol 119:427-42
Zhang, Ping; Canessa, Cecilia M (2002) Single channel properties of rat acid-sensitive ion channel-1alpha, -2a, and -3 expressed in Xenopus oocytes. J Gen Physiol 120:553-66

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