Biomedical subjects

O Al-Khalili

Publications and source records attributed to O Al-Khalili.

3 recordsLinked to original sources

PubMed2001

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway.

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.

Amino Acid Sequence↗

PubMed1999

Isoprenylcysteine-O-carboxyl methyltransferase regulates aldosterone-sensitive Na(+) reabsorption.

The Xenopus laevis distal tubule epithelial cell line A6 was used as a model epithelia to study the role of isoprenylcysteine-O-carboxyl methyltransferase (pcMTase) in aldosterone-mediated stimulation of Na(+) transport. Polyclonal antibodies raised against X. laevis pcMTase were immunoreactive with a 33-kDa protein in whole cell lysate. These antibodies were also reactive with a 33-kDa product from in vitro translation of the pcMTase cDNA. Aldosterone application increased pcMTase activity resulting in elevation of total protein methyl esterification in vivo, but pcMTase protein levels were not affected by steroid, suggesting that aldosterone increased activity independent of enzyme number. Inhibition of pcMTase resulted in a reduction of aldosterone-induced Na(+) transport demonstrating the necessity of pcMTase-mediated transmethylation for steroid induced Na(+) reabsorption. Transfection with an eukaryotic expression construct containing pcMTase cDNA increased pcMTase protein level and activity. This resulted in potentiation of the natriferic actions of aldosterone. However, overexpression did not change Na(+) reabsorption in the absence of steroid, suggesting that pcMTase activity is not limiting Na(+) transport in the absence of steroid, but that subsequent to aldosterone addition, pcMTase activity becomes limiting. These results suggest that a critical transmethylation is necessary for aldosterone-induction of Na(+) transport. It is likely that the protein catalyzing this methylation is isoprenylcysteine-O-carboxyl methyltransferase and that aldosterone activates pcMTase without affecting transferase expression.

Absorption↗

PubMed1999

Antisense oligonucleotides against the alpha-subunit of ENaC decrease lung epithelial cation-channel activity.

Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+ transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits alpha, beta, and gamma in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 +/- 1.1 pS and an Na+-to-K+ selectivity of 0.97 +/- 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (alpha, beta, and gamma) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the alpha-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the beta- and gamma-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the alpha-subunit of ENaC alone or in combination with some protein other than the beta- or gamma-subunit protein is the major component of lung alveolar epithelial cation channels.

Animals↗