The Epithelial Na+ Channel or ENaC is rate limiting to sodium absorption in renal epithelia. Channel activity sets the final urinary sodium concentration and determines in part sodium excretion. Channel activity affects body sodium and water balance and therefore blood pressure. This channel exists at the plasma membrane largely in an electrically inactive pool. Recently, it was demonstrated that this inactive pool can be activated by cleavage of two of the channel subunits by serine proteases. Thus, this mode of channel regulation affords ENaC expressing renal epithelia an enormous capacity to rapidly respond to changes of sodium load, renal filtration, and body sodium balance. Activation of silent channels by proteases is proposed to involve the removal of short inhibitory domains from the channel's alpha and gamma subunits. However, our data clearly demonstrate that non-cleaved channels can be active, while some cleaved channel subunits do not form active channels. We demonstrate that Po can be regulated by multiple mechanisms that include channel processing, membrane lipid rigidity, channel partitioning into lipid rafts, and interaction between inhibitory domains of the ENaC subunits. We propose that multiple interactions between the ENaC subunits determine channel Po. Cleavage of the channel subunits provides a mean of removing or altering some of these interactions and leads to channel activation. We also propose that channel subunit interactions affect subunit processing, membrane partitioning and stability. The unifying hypothesis is that unprocessed channels are stable at the membrane but inherently inactive unless conditions exist to reduce subunit-subunit inhibition, and that moreover, proteolysis activates or primes the channels for activation by permanently removing such inter-subunit interactions. Proteolysis then predisposes the channel subunits for internalization unless processed subunits are protected in raft membrane domains. To address the mechanism of control of channel activity, we develop new tools that utilize 1) engineered cleavage sites 2) a recently identified constitutively ENaC subunit (epsilon) 3) homology mapping of the channel to the crystal structure of ASIC1 and 4) analysis of channel partitioning into membrane domains. Our data will pave the way for understanding channel Po regulation and every downstream process that further modify this Po.

Public Health Relevance

We examine the mechanisms that control the spontaneous activity of the renal epithelial sodium channel. The activity of this protein is important in determining kidney salt excretion and consequently salt retention by the body and blood volume and pressure. Understanding the spontaneous activity of this protein would help understand the basis of salt sensitivity of blood pressure and is important in designing therapy for individuals with high blood pressure.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Cellular and Molecular Biology of the Kidney Study Section (CMBK)
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Ketchum, Christian J
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State University of New York at Buffalo
Schools of Medicine
United States
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Berman, Jonathan M; Awayda, Ryan G; Awayda, Mouhamed S (2015) Interacting domains in the epithelial sodium channel that mediate proteolytic activation. Channels (Austin) 9:281-90
Berman, Jonathan M; Brand, Cristin; Awayda, Mouhamed S (2015) A long isoform of the epithelial sodium channel alpha subunit forms a highly active channel. Channels (Austin) 9:30-43
Berman, Jonathan M; Awayda, Mouhamed S (2013) Redox artifacts in electrophysiological recordings. Am J Physiol Cell Physiol 304:C604-13
Gondzik, Veronika; Weber, Wolf Michael; Awayda, Mouhamed S (2012) Coupling of epithelial Na+ and Cl- channels by direct and indirect activation by serine proteases. Am J Physiol Cell Physiol 303:C936-46
Awayda, Mouhamed S; Awayda, Karen L; Pochynyuk, Oleh et al. (2011) Acute cholesterol-induced anti-natriuretic effects: role of epithelial Na+ channel activity, protein levels, and processing. J Biol Chem 286:1683-95
Gondzik, Veronika; Awayda, Mouhamed S (2011) Methods for stable recording of short-circuit current in a Na+-transporting epithelium. Am J Physiol Cell Physiol 301:C162-70
Hu, John Cong; Bengrine, Abderrahmane; Lis, Agnieszka et al. (2009) Alternative mechanism of activation of the epithelial na+ channel by cleavage. J Biol Chem 284:36334-45
Ortiz, Rudy M; Graciano, Miguel L; Seth, Dale et al. (2007) Aldosterone receptor antagonism exacerbates intrarenal angiotensin II augmentation in ANG II-dependent hypertension. Am J Physiol Renal Physiol 293:F139-47
Bengrine, Abderrahmane; Li, Jinqing; Hamm, L Lee et al. (2007) Indirect activation of the epithelial Na+ channel by trypsin. J Biol Chem 282:26884-96
Bengrine, Abderrahmane; Li, Jinqing; Awayda, Mouhamed S (2007) The A-kinase anchoring protein 15 regulates feedback inhibition of the epithelial Na+ channel. FASEB J 21:1189-201

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