This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Under conditions of metabolic stress, the expression and activity of many membrane transport proteins including the epithelial sodium channel, ENaC, are inhibited, thereby limiting the dissipation of ionic gradients and preserving the cellular energy required to maintain them. The cellular mechanisms that link membrane transport to energy production and metabolic status are only beginning to be revealed. The authors have previously shown that ENaC is inhibited by AMP-activated protein kinase (AMPK, a ubiquitous serine/theonine kinase that participates as a metabolic sensor and metabolic regulator in many systems). AMPK does not directly phosphorylate ENaC, but may interact with regulators of ENaC-mediated Na+ transport such as the ubiquitin ligase, Nedd4-2. Preliminary experiments indicate that AMPK is unable to inhibit ENaC-mediated Na+ current when using variants of ENaC subunits unable to interact with Nedd4-2. Additionally, AMPK phosphorylates Nedd4-2 in vitro. This line of evidence suggests the following hypothesis: Nedd4-2 may serve as a direct target of AMPK, and AMPK may phosphorylate Nedd4-2 and therefore augment its ability to bind to ENaC or its ubiquitin ligase activity. In order to test this hypothesis, the authors need to establish whether AMPK phosphorylates Nedd4-2 in vivo and at what sites this phosphorylation occurs. We will modulate AMPK activity in cultured cells and purify Nedd4-2 for analysis. We will analyze sites of AMPK phosphorylation of Nedd4-2 by strong cation exchange chromatography fractionation of a tryptic digest of Nedd4-2 followed by non-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry.
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