Biomedical subjects

Rachell E Booth

Publications and source records attributed to Rachell E Booth.

4 recordsLinked to original sources

PubMed2003

A region directly following the second transmembrane domain in gamma ENaC is required for normal channel gating.

We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse alpha, beta, and gamma epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of alphaENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, gammaENaC contained such a domain just distal to TM2 spanning Gln573-Pro600. Interactive domains were also localized within Met1-Gln54 and the last 17 residues of alpha- and betaENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH2 terminus of alphaENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of gammaENaC was established with whole-cell patch clamp experiments of wild type (alpha, beta, and gamma) and mutant (alpha, beta, and gammadeltaQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 +/- 0.14 versus 2.5 +/- 0.80 nA of amiloridesensitive inward current at -80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in Po with 0.16 +/- 0.03 versus 0.67 +/- 0.07 for wild type. Mutant gammaENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln573-Pro600, deletion of Gln573-Arg583 but not Thr584-Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573-Arg583 of gammaENaC are necessary for normal channel gating.

Amino Acid Motifs↗

PubMed2003

Targeted degradation of ENaC in response to PKC activation of the ERK1/2 cascade.

Renal A6 epithelial cells were used to determine the mechanism by which protein kinase C (PKC) decreases epithelial Na(+) channel (ENaC) activity. Activation of PKC reduced relative Na(+) reabsorption to <20% within 60 min. This decrease was sustained over the next 24-48 h. In response to PKC signaling, alpha-, beta-, and gamma-ENaC levels were 0.97, 0.36, and 0.39, respectively, after 24 h, with the levels of the latter two subunits being significantly decreased. The PKC-mediated decreases in beta- and gamma-ENaC were significantly reversed by simultaneous addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1/2 inhibitors U-0126 and PD-98059. These inhibitors, in addition, protected Na(+) reabsorption from PKC, demonstrating that the MAPK1/2 cascade, in some instances, plays a central role in downregulation of ENaC activity. The effects of PKC on beta- and gamma-ENaC levels were additive with those of inhibitors of transcription (actinomycin D) and translation (emetine and cycloheximide), suggesting that PKC promotes subunit degradation and does not affect subunit synthesis. The bulk of whole cell gamma-ENaC was degraded within 1 h after treatment with inhibitors of synthesis; however, a significant pool was "protected" from inhibitors for up to 12 h. PKC affected this protected pool of gamma-ENaC. Moreover, proteosome inhibitors (MG-132 and lactacystin) reversed PKC effects on this protected pool of gamma-ENaC. Thus PKC signaling via MAPK1/2 cascade activation in A6 cells promotes degradation of gamma-ENaC.

Animals↗

PubMed2002

Identification of cytoplasmic domains within the epithelial Na+ channel reactive at the plasma membrane.

The activity of membrane proteins is controlled, in part, by protein-protein interactions localized to the plasma membrane. In the current study, domains within the epithelial Na(+) channel (ENaC) reactive at the plasma membrane were identified using a novel yeast one-hybrid screen. The cytosolic N terminus of alphaENaC and the cytosolic C termini of alpha-, beta-, and gammaENaC contained domains reactive at the plasma membrane. Fluorescent micrographs of epithelial cells overexpressing fusion proteins of enhanced green fluorescent protein and mENaC cytosolic domains were consistent with those in yeast. A novel membrane reactive domain within the cytosolic C terminus of gamma-mENaC was localized to the 17 amino acids between residues Thr(584)-Pro(600). Two overlapping internalization signals within the C terminus of gamma-mENaC, a WW-binding domain (PY motif) and a tyrosine-based endocytic signal, were additive with respect to decreasing complementation and expression levels of hybrid proteins. Decreases in expression levels of hybrid proteins containing the PY and endocytic motif were reversed with latrunculin A, an inhibitor of endosomal movement. Decreases in complementation and expression levels of hybrid proteins mediated by the combined PY and overlapping endocytic motif proceeded in the absence of established ubiquitination sites within ENaC. In addition, the endocytic motif was active in the absence of the PY motif, demonstrating that these two domains, while possibly interacting, also have discrete functions. The novel domains within the cytosolic N terminus of alphaENaC and the C termini of alpha-, beta-, and gammaENaC identified here are likely to be involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane. We hypothesize that these newly identified domains play a role in modulating ENaC activity.

Amino Acid Motifs↗

PubMed2002

Aldosterone.

Aldosterone plays a pivotal role in electrolyte and fluid homeostasis and thus control of blood pressure. The "classical" view of aldosterone action is that it targets epithelia of the distal colon and renal nephron to stimulate Na(+) (re)absorption and K(+) secretion. In these cells, aldosterone binds steroid receptors, promoting translocation to the nucleus, where they modulate gene expression with the induced proteins stimulating transport. This "genomic" action is dependent on transcription and translation and has a latency of 0.5-1.0 h. Recently, more rapid actions of aldosterone that are independent of transcription and translation have been described. These "nongenomic" actions are mediated by a distinct receptor that is insensitive to inhibitors of the classical mineralocorticoid receptor, such as spironolactone. The present review describes advances in our understanding of the classical model of aldosterone action as well as those that broaden this model to encompass nongenomic actions, nonepithelial targets, production of aldosterone outside of the adrenal gland, novel mechanisms of specificity, and novel mechanisms for mediating genomic actions.

Aldosterone↗