The epithelial sodium channel (ENaC) is composed of three homologous subunits termed ␣, ␤, and ␥. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the ␣-subunit to cysteine and co-expressing these mutants with wild type ␤-and ␥-subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (␣-Val 569 -␣-Gly 579 ) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (␣-Ser 580 -␣-Ser 592 ) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing ␣G587C or ␣S589C were permeable to K ؉ , and mutation of a GSS tract (positions ␣587-␣589) to GYG resulted in a moderately K ؉ -selective channel. Our results suggest that the C-terminal portion of the pore region within the ␣-subunit contributes to the selectivity filter of ENaC.Epithelial sodium channels (ENaCs) 1 mediate sodium transport across apical plasma membranes of epithelial cells that line the distal nephron, the airway and alveoli, and the distal colon. ENaCs are composed of three homologous subunits, termed ␣-, ␤-, and ␥ENaC (1, 2) and have a subunit stoichiometry of ␣2:␤1:␥1 (3, 4) ( Fig. 1B), although some reports have suggested an alternative subunit stoichiometry (5, 6). The three subunits have a similar topology, with cytoplasmic amino and carboxyl termini and two transmembrane domains (termed M1 and M2) that are separated by a large ectodomain (Fig. 1A) (7-9). Hydropathy analyses revealed two rather hydrophobic regions (termed H1 and H2) immediately following M1 and preceding M2 (2). The region preceding M2 may enter the membrane, similar to the pore-forming region (P region) of the voltage-gated Na ϩ , K ϩ , and Ca 2ϩ channels (9).Mutagenesis of selected residues within the H2 region and the segment between H2 and M2 of the ␣-, ␤-, or ␥-subunits of ENaC resulted in changes in cation selectivity, single channel conductance, or sensitivity to amiloride, a putative pore blocker of ENaC (4, 10 -13). These findings argued that all three subunits are involved in pore formation and that the region preceding the M2 domain forms a portion of the pore. Snyder et al. (14) recently reported a study describing the results of scanning mutagenesis of the pore region of the ␥-subunit of human ENaC. Both Snyder et al. and Kellenberger et al. (11,12,14) have suggested that the pore structure of ENaC is distinct from that of KcsA, a K ϩ channel whose structure was resolved by x-ray c...

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydrylreactive methanethiosulfonate derivatives ( (2- , whereas several other mutant channels were partially blocked by MTSEA or Cd 2؉ but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an ␣-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.Epithelial sodium channels (ENaCs) 1 are composed of three homologous subunits, termed ␣-, ␤-, and ␥ENaC (1, 2). These subunits assemble to form a hetero-oligomeric, Na ϩ -selective ion channel with a subunit stoichiometry of 2␣:1␤:1␥ (3, 4), although an alternative subunit stoichiometry has been proposed (5, 6). All three Na ϩ channel subunits have cytoplasmic amino and carboxyl termini, two transmembrane domains (termed M1 and M2), and a large ectodomain (7-9). Previous studies have shown that selected point mutations within the pore region preceding M2 of each subunit altered functional properties of the channel, including cation selectivity, single channel conductance, and sensitivity to the blocker amiloride (4, 10 -14). Specific mutations of residues in a conserved threeresidue tract, (G/S)XS (where X is Ser, Gly, or Cys), within the pore region of the three ENaC subunits, rendered channels K ϩ -permeable. Snyder et al. (15) examined the accessibility of a sulfhydryl-reactive methane thiosulfonate (MTS) derivative to substituted cysteine residues within the pore region of human ␥ENaC, and they proposed a structural model of the channel pore similar to that proposed by Kellenberger et al. (11) but distinct from the resolved structure of the KcsA K ϩ channel pore (16).We previously reported that selected cysteine substitutions within the carboxyl-terminal domain of the pore region of mouse ␣ENaC (␣Ser 580 -␣Ser 592 ) altered the cation selectivity and amiloride sensitivity of the channel and proposed that this region forms the selectivity filter of the channel (14). In the current study, we systematically examined accessibility of sulfhydryl reagents to ␣␤␥mENaCs with engineered cysteine within the 24-residue pore region of the ␣-subunit. Channels with selected cysteine mutations within the carboxyl-terminal portion of the pore region responded to the external application of MTS derivatives with an inhibition of amiloride-sensitive Na ϩ currents. In contrast, we observed a significant increase in amiloride-sensitive Na ϩ currents following the external application of MTS derivatives or Cd 2ϩ when cysteine residues were introduced at selected sites within the amino-terminal portion of the pore region of ␣mENaC. The pattern of distribution of cysteine mutations that led to MTS-induced activation of Na ϩ currents suggests that this region has an ␣-helical structure. In addition, the activation of ␣S580C␤␥ by an MTS reage...

Novel urinary kidney safety biomarkers have been identified recently that may outperform or add value to the conventional renal function biomarkers, blood urea nitrogen (BUN) and serum creatinine (SCr). To assess the relative performance of the growing list of novel biomarkers, a comprehensive evaluation was conducted for 12 urinary biomarkers in 22 rat studies including 12 kidney toxicants and 10 compounds with toxicities observed in organs other than kidney. The kidney toxicity studies included kidney tubular toxicants and glomerular toxicants. The 12 urinary biomarkers evaluated included Kim-1, clusterin, osteopontin, osteoactivin, albumin, lipocalin-2, GST-α, β2-microglobulin, cystatin C, retinol binding protein 4, total protein, and N-acetyl-β-D-glucosaminidase. Receiver operator characteristic (ROC) curves were generated for each biomarker and for BUN and SCr to compare the relative performance of the 12 biomarkers in individual animals against the microscopic histomorphologic changes observed in the kidney. Among the kidney toxicity biomarkers analyzed, Kim-1, clusterin, and albumin showed the highest overall performance for detecting drug-induced renal tubular injury in the rat in a sensitive and specific manner, whereas albumin showed the highest performance in detecting drug-induced glomerular injury. Although most of the evaluated kidney biomarkers were more sensitive in detecting kidney toxicity compared with BUN and SCr, all biomarkers demonstrated some lack of specificity, most notably NGAL and osteopontin, illustrating the need for caution when interpreting urinary biomarker increases in rat samples when organ toxicity is unknown.

The mammalian urinary bladder exhibits transepithelial Na+ absorption that contributes to Na+ gradients established by the kidney. Electrophysiological studies have demonstrated that electrogenic Na+ absorption across the urinary bladder is mediated in part by amiloride-sensitive Na+ channels situated within the apical membrane of the bladder epithelium. We have used a combination of in situ hybridization, Northern blot analysis, and immunocytochemistry to examine whether the recently cloned epithelial Na+ channel (ENaC) is expressed in the rat urinary bladder. In situ hybridization and Northern blot analyses indicate that α-, β-, and γ-rat ENaC (rENaC) are expressed in rat urinary bladder epithelial cells. Quantitation of the levels of α-, β-, and γ-rENaC mRNA expression in rat urinary bladder, relative to β-actin mRNA expression, indicates that, although comparable levels of α- and β-rENaC subunits are expressed in the urinary bladder of rats maintained on standard chow, the level of γ-rENaC mRNA expression is 5- to 10-fold lower than α- or β-rENaC mRNA. Immunocytochemistry, using an antibody directed against α-rENaC, revealed that ENaCs are predominantly localized to the luminal membrane of the bladder epithelium. Together, these data demonstrate that ENaC is expressed in the mammalian urinary bladder and suggest that amiloride-sensitive Na+ transport across the apical membrane of the mammalian urinary bladder epithelium is mediated primarily by ENaC.

Rodents, mice and rats in particular, are the species of choice for evaluating chemical carcinogenesis. However, different species and strains often respond very differently, undermining the logic of extrapolation of animal results to humans and complicating risk assessment. Intracisternal A particles (IAPs), endogenous retroviral sequences, are an important class of transposable elements that induce genomic mutations and cell transformation by disrupting gene expression. Several lines of evidence support a role of IAPs as mouse-specific genetic factors in responses to toxicity and expression of disease phenotypes. Since multiple subtypes and copies of IAPs are present in the mouse genome, their activity and locations relative to functional genes are of critical importance. This study identified the major "active" subtypes of IAPs (subtype 1/1a) that are responsible for newly transposed IAP insertions described in the literature, and confirmed that (1) polymorphisms for IAP insertions exist among different mouse strains and (2) promoter activity of the LTRs can be modulated by chemicals. This study further identified all the genes in the C57BL/6 mouse genome with IAP subtype 1 and 1a sequences inserted in their proximity, and the major biofunctional categories and cellular signaling networks of those genes. Since many "IAP-associated genes" play important roles in the regulation of cell proliferation, cell cycle, and cell death, the associated IAPs, upon activation, can affect cellular responses to xenobiotics and disease processes, especially carcinogenesis. This systemic analysis provides a solid foundation for further investigations of the role of IAPs as species- and strain-specific disease susceptibility factors.