(1/1112) Tetraethylammonium block of the BNC1 channel.
The brain Na+ channel-1 (BNC1, also known as MDEG1 or ASIC2) is a member of the DEG/ENaC cation channel family. Mutation of a specific residue (Gly430) that lies N-terminal to the second membrane-spanning domain activates BNC1 and converts it from a Na+-selective channel to one permeable to both Na+ and K+. Because all K+ channels are blocked by tetraethylammonium (TEA), we asked if TEA would inhibit BNC1 with a mutation at residue 430. External TEA blocked BNC1 when residue 430 was a Val or a Thr. Block was steeply voltage-dependent and was reduced when current was outward, suggesting multi-ion block within the channel pore. Block was dependent on the size of the quaternary ammonium; the smaller tetramethylammonium blocked with similar properties, whereas the larger tetrapropylammonium had little effect. When residue 430 was Phe, the effects of tetramethylammonium and tetrapropylammonium were not altered. In contrast, block by TEA was much less voltage-dependent, suggesting that the Phe mutation introduced a new TEA binding site located approximately 30% of the way across the electric field. These results provide insight into the structure and function of BNC1 and suggest that TEA may be a useful tool to probe function of this channel family. (+info)
(2/1112) Epithelial sodium channel regulated by aldosterone-induced protein sgk.
Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na+ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na+ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na+ absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis. (+info)
(3/1112) Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes.
1. Regulation of the amiloride-sensitive epithelial sodium channel (ENaC) is essential for the control of body sodium homeostasis. The downregulation of the activity of this Na+ channel that occurs when the intracellular Na+ concentration ([Na+]i) is increased is known as feedback inhibition. Although intracellular Na+ is the trigger for this phenomenon, its cellular and molecular mediators are unknown. 2. We used the 'cut-open oocyte' technique to control the composition of the intracellular milieu of Xenopus oocytes expressing rat ENaCs to enable us to test several factors potentially involved in feedback inhibition. 3. The effects of perfusion of the intracellular space were demonstrated by an electromicrographic study and the time course of the intracellular solution exchange was established by observing the effect of intracellular pH: a decrease from pH 7.4 to 6.5 reduced the amiloride-sensitive current by about 40 % within 2 min. 4. Feedback inhibition was observed in non-perfused oocytes when Na+ entry induced a large increase in [Na+]i. Intracellular perfusion prevented feedback regulation even though the [Na+]i was allowed to increase to values above 50 mM. 5. No effects on the amiloride-sensitive current were observed after changes in the concentration of Na+ (from 1 to 50 mM), Ca2+ (from 10 to 1000 nM) or ATP (from nominally free to 1 or 5 mM) in the intracellular perfusate. 6. We conclude that feedback inhibition requires intracellular factors that can be removed by intracellular perfusion. Although a rise in [Na+]i may be the trigger for the feedback inhibition of the ENaC, this effect is not mediated by a direct effect of Na+, Ca2+ or ATP on the ENaC protein. (+info)
(4/1112) Genetic disorders of membrane transport. V. The epithelial sodium channel and its implication in human diseases.
The epithelial Na+ channel (ENaC) controls the rate-limiting step in the process of transepithelial Na+ reabsorption in the distal nephron, the distal colon, and the airways. Hereditary salt-losing syndromes have been ascribed to loss of function mutations in the alpha-, beta-, or gamma-ENaC subunit genes, whereas gain of function mutations (located in the COOH terminus of the beta- or gamma-subunit) result in hypertension due to Na+ retention (Liddle's syndrome). In mice, gene-targeting experiments have shown that, in addition to the kidney salt-wasting phenotype, ENaC was essential for lung fluid clearance in newborn mice. Disruption of the alpha-subunit resulted in a complete abolition of ENaC-mediated Na+ transport, whereas knockout of the beta- or gamma-subunit had only minor effects on fluid clearance in lung. Disruption of each of the three subunits resulted in a salt-wasting syndrome similar to that observed in humans. (+info)
(5/1112) Developmental expression of sodium entry pathways in rat nephron.
During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11beta-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins. (+info)
(6/1112) Antiidiotypic antibody recognizes an amiloride binding domain within the alpha subunit of the epithelial Na+ channel.
We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alphaENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alphaENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alphaENaC. (+info)
(7/1112) A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving.
The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC beta and gamma subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC alpha subunit (alphaS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the alphaS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that alphaS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration. (+info)
(8/1112) The pre-transmembrane 1 domain of acid-sensing ion channels participates in the ion pore.
The acid-sensing ion channel (ASIC) subunits ASIC1, ASIC2, and ASIC3 are members of the amiloride-sensitive Na+ channel/degenerin family of ion channels. They form proton-gated channels that are expressed in the central nervous system and in sensory neurons, where they are thought to play an important role in pain accompanying tissue acidosis. A splice variant of ASIC2, ASIC2b, is not active on its own but modifies the properties of ASIC3. In particular, whereas most members of the amiloride-sensitive Na+ channel/degenerin family are highly selective for Na+ over K+, ASIC3/ASIC2b heteromultimers show a nonselective component. Chimeras of the two splice variants allowed identification of a 9-amino acid region preceding the first transmembrane (TM) domain (pre-TM1) of ASIC2 that is involved in ion permeation and is critical for Na+ selectivity. Three amino acids in this region (Ile-19, Phe-20, and Thr-25) appear to be particularly important, because channels mutated at these residues discriminate poorly between Na+ and K+. In addition, the pH dependences of the activity of the F20S and T25K mutants are changed as compared with that of wild-type ASIC2. A corresponding ASIC3 mutant (T26K) also has modified Na+ selectivity. Our results suggest that the pre-TM1 region of ASICs participates in the ion pore. (+info)