The epithelial Na channel (ENaC) plays a fundamental role in establishing blood pressure. ENaC activity is set, in part, by its level in the plasma membrane. Membrane levels of ENaC reflect constitutive delivery and regulated retrieval. The mechanisms and domains within ENaC involved in retrieval are not completely understood. We are interested in two conserved, overlying domains (S/TPPPxYxS/TL) found within all ENaC subunits: the PY (xPPxY) and tyrosine-based endocytic (YxxL) motifs. The prior motif targets the channel for ubiquitinylation via Nedd4 ubiquitin ligases; and the latter motif is known to interact with the mu2 subunit of the AP-2 complex, which targets proteins for clathrin coated-pit endocytosis mediated by dynamin. We will test whether these domains are modular and thus, separable or whether they act in concert. Physiological signaling cascades (e.g. MAPK) decrease ENaC activity by promoting channel degradation. Thus, we also test the hypothesis that MAPK signaling decreases channel activity via these modular retrieval domains. Moreover, we will determine whether absolutely conserved S/T just preceding the PY and within the YxxL motifs, which fit the consensus sequence for MAPK, are targets for MAPK impacting channel activity and membrane level. ENaC is a heteromeric channel comprised of 3 distinct subunits with channels containing two or fewer types of subunits having decreased activity. Thus, one possible outcome of subunit retrieval is production of homomeric channels or channels containing only two types of subunits. Since, subunit stoichiometry of membrane ENaC may not be fixed, we also ask whether MAPK signaling via the PY and YxxL domains changes ENaC subunit stoichiometry and/or composition to modulate channel activity. Here, we address four specific aims: 1) Determine subunit stoichiometry of membrane ENaC; 2) Determine whether membrane ENaC levels are controlled by modular PY and YxxL endocytic domains and assign significance to each domain; 3) Determine whether physiological cell signaling cascades modulate channel retrieval via the PY and YxxL motifs and whether differential phosphorylation of conserved S/T modulate this retrieval; and 4) Determine if retrieval of ENaC subunits from the plasma membrane is coordinated. This research will provide critical insight about the molecualr architecture of ENaC and regulation of this important channel.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK070571-03
Application #
7390345
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ketchum, Christian J
Project Start
2006-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2009-03-31
Support Year
3
Fiscal Year
2008
Total Cost
$227,846
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Physiology
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Mironova, Elena; Bugay, Vladislav; Pochynyuk, Oleh et al. (2013) Recording ion channels in isolated, split-opened tubules. Methods Mol Biol 998:341-53
Toney, Glenn M; Vallon, Volker; Stockand, James D (2012) Intrinsic control of sodium excretion in the distal nephron by inhibitory purinergic regulation of the epithelial Na(+) channel. Curr Opin Nephrol Hypertens 21:52-60
Kucher, Volodymyr; Boiko, Nina; Pochynyuk, Oleh et al. (2011) Voltage-dependent gating underlies loss of ENaC function in Pseudohypoaldosteronism type 1. Biophys J 100:1930-9
Mironova, Elena; Stockand, James D (2010) Activation of a latent nuclear localization signal in the NH2 terminus of ?-ENaC initiates feedback regulation of channel activity. Am J Physiol Renal Physiol 298:F1188-96
Pochynyuk, Oleh; Kucher, Volodymyr; Boiko, Nina et al. (2009) Intrinsic voltage dependence of the epithelial Na+ channel is masked by a conserved transmembrane domain tryptophan. J Biol Chem 284:25512-21
Dorofeeva, Natalia A; Karpushev, Alexey V; Nikolaev, Maxim V et al. (2009) Muscarinic M1 modulation of acid-sensing ion channels. Neuroreport 20:1386-91
Bugaj, Vladislav; Pochynyuk, Oleh; Mironova, Elena et al. (2008) Regulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct. Am J Physiol Renal Physiol 295:F1063-70
Stockand, James D; Staruschenko, Alexander; Pochynyuk, Oleh et al. (2008) Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure. IUBMB Life 60:620-8
Pochynyuk, Oleh; Bugaj, Vladislav; Rieg, Timo et al. (2008) Paracrine regulation of the epithelial Na+ channel in the mammalian collecting duct by purinergic P2Y2 receptor tone. J Biol Chem 283:36599-607
Pochynyuk, Oleh; Bugaj, Vladislav; Vandewalle, Alain et al. (2008) Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells. Am J Physiol Renal Physiol 294:F38-46

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