Biomedical subjects

James D Stockand

Publications and source records attributed to James D Stockand.

8 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↗

PubMed2003

Subunit-specific modulation of KCNQ potassium channels by Src tyrosine kinase.

We studied regulation by c-Src tyrosine kinase (Src) of KCNQ1-5 channels heterologously expressed in Chinese hamster ovary (CHO) cells and of native M current in rat sympathetic neurons. Using whole-cell patch clamp, we found that Src modulates currents from KCNQ3, KCNQ4, and KCNQ5 homomultimers, KCNQ2/3 heteromultimers and native M current, but not currents from KCNQ1 or KCNQ2 homomultimers. Src overexpression had two effects: a decrease of current amplitude (4- to 15-fold for cloned channels and approximately 3-fold for M current) and a slowing of activation kinetics by 2-fold. Both Src actions were mostly reversed by bath application of the Src inhibitors erbstatin (20 microm) and PP2 (200 nm), and mimicked by the tyrosine phosphatase inhibitor sodium vanadate (100 microm). Immunoprecipitation and immunoblot analysis showed Src-dependent phosphotyrosine signals associated with KCNQ3, KCNQ4, and KCNQ5 but not with KCNQ1 or KCNQ2 that may be tyrosine phosphorylation of the channel subunits. Expression of a dominant negative Src that cannot phosphorylate substrates had no effect on the current and did not induce phosphotyrosine signals associated with KCNQ3-5 subunits, further indicating that Src actions on KCNQ currents are mediated by tyrosine phosphorylation. Immunostaining and confocal analysis showed no effect of Src overexpression on the abundance of KCNQ3 protein in CHO cells. Finally, experiments using cloned KCNQ2/3 channels, Src and M(1) muscarinic receptors, and sympathetic neurons demonstrated that the actions on KCNQ channels by Src and by muscarinic agonists use distinct mechanisms.

Animals↗

PubMed2002

Aldosterone stimulates proliferation of cardiac fibroblasts by activating Ki-RasA and MAPK1/2 signaling.

Aldosterone plays a pathological role in cardiac fibrosis by directly affecting cardiac fibroblasts. Understanding of the cellular mechanisms of aldosterone action in cardiac fibroblasts, however, is rudimentary. One possibility is that aldosterone promotes proliferation of cardiac fibroblasts by activating specific cellular signaling cascades. The current study tests whether aldosterone stimulates proliferation of isolated adult rat cardiac myofibroblasts (RCF) by activating Kirsten Ras (Ki-RasA) and its effector, the MAPK1/2 cascade. Aldosterone (10 nM) significantly increased RCF proliferation. This action was sensitive to the mineralocorticoid receptor (MR) antagonist spironolactone. Expression of MR in RCF and the whole rat heart was confirmed by immunoblotting. Aldosterone significantly increased absolute and active (GTP bound) Ki-RasA levels in RCF. Aldosterone, in addition, significantly increased phospho-c-Raf and phospho-MAPK1/2. The effects of aldosterone on Ki-RasA and phospho-c-Raf proteins were inhibited by spironolactone but not RU-486, suggesting that aldosterone acts via MR. Inhibitors of MEK1/2 and c-Raf prevented aldosterone-induced activation of MAPK1/2 and proliferation. These results show that aldosterone directly increases RCF proliferation through MR-dependent activation of Ki-RasA and its effector, the MAPK1/2 cascade. Activation of cardiac fibroblasts through such a cascade may play a role in the pathological actions exerted by aldosterone on the heart.

Aldosterone↗

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

Activation of mitogen-activated protein kinase (mitogen-activated protein kinase/extracellular signal-regulated kinase) cascade by aldosterone.

Aldosterone in some tissues increases expression of the mRNA encoding the small monomeric G protein Ki-RasA. Renal A6 epithelial cells were used to determine whether induction of Ki-ras leads to concomitant increases in the total as well as active levels of Ki-RasA and whether this then leads to subsequent activation of its effector mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase) cascade. The molecular basis and cellular consequences of this action were specifically investigated. We identified the intron 1-exon 1 region (rasI/E1) of the mouse Ki-ras gene as sufficient to reconstitute aldosterone responsiveness to a heterologous promotor. Aldosterone increased reporter gene activity containing rasI/E1 threefold. Aldosterone increased the absolute and GTP-bound levels of Ki-RasA by a similar extent, suggesting that activation resulted from mass action and not effects on GTP binding/hydrolysis rates. Aldosterone significantly increased Ki-RasA and MAPK activity as early as 15 min with activation peaking by 2 h and waning after 4 h. Inhibitors of transcription, translation, and a glucocorticoid receptor antagonist attenuated MAPK signaling. Similarly, rasI/E1-driven luciferase expression was sensitive to glucocorticoid receptor blockade. Overexpression of dominant-negative RasN17, addition of antisense Ki-rasA and inhibition of mitogen-activated protein kinase kinase also attenuated steroid-dependent increases in MAPK signaling. Thus, activation of MAPK by aldosterone is dependent, in part, on a genomic mechanism involving induction of Ki-ras transcription and subsequent activation of its downstream effectors. This genomic mechanism has a distinct time course from activation by traditional mitogens, such as serum, which affect the GTP-binding state and not absolute levels of Ras. The result of such a genomic mechanism is that peak activation of the MAPK cascade by adrenal corticosteroids is delayed but prolonged.

Aldosterone↗

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↗

PubMed2002

New ideas about aldosterone signaling in epithelia.

The systemic actions of aldosterone are well documented; however, in comparison, our understanding of the cellular and molecular mechanisms by which aldosterone orchestrates these actions is rudimentary. Aldosterone exerts most of its physiological actions by modifying gene expression. It is now apparent that aldosterone represses almost as many genes as it induces. Several aldosterone-sensitive genes, including serum and glucocorticoid-inducible kinase (sgk) and small, monomeric Kirsten Ras GTP-binding protein (Ki-ras) have recently been identified. The molecular mechanisms and elements bestowing corticosteroid sensitivity on these and many other genes are becoming clear. Induction of Ki-Ras and Sgk is necessary and sufficient for some portion of aldosterone action in epithelia. These two signaling factors are components of a converging pathway with phosphatidylinositol 3-kinase positioned between them that enables both stabilizing the epithelial Na(+) channel (ENaC) in the open state as well as increasing the number of ENaC in the apical membrane. This aldosterone-induced signaling pathway contains many potential sites for feedback regulation and cross talk from other cascades and potentially impinges directly on the activity of transport proteins and/or cellular differentiation to modify electrolyte transport.

Aldosterone↗