Vadim Shlyonsky

Identification of an Amiloride Binding Domain within the α-Subunit of the Epithelial Na+ Channel

Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278–283) within the extracellular domain of αrENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant αrENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type αrENaC was inhibited by amiloride with aK i of 169 nm. Deletion of the entire WYRFHY tract (αrENaC Δ278–283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (K i = 26.5 μm). Similar results were obtained when either αrENaC or αrENaC Δ278–283 were co-expressed with wt β- and γrENaC (K i values of 155 nm and 22.8 μm, respectively). Moreover, αrENaC H282D was insensitive to submicromolar concentrations of amiloride (K i = 6.52 μm), whereas αrENaC H282R was inhibited by amiloride with a K i of 29 nm. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of αrENaC and mutations preceding the second membrane-spanning domains of α-, β-, and γrENaC alters amiloride’s K i , suggest that selected regions of the extracellular loop of αrENaC may be in close proximity to residues within the channel pore.

Carboxylmethylation of the beta subunit of xENaC regulates channel activity.

The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. Aldosterone stimulates methylation of a 95-kDa protein in apical membrane of A6 cells, and we have previously shown that methylation of a 95-kDa protein in the immunopurified Na+ channel complex increases open probability of these channels in planar lipid bilayers. We report here that aldosterone stimulates carboxylmethylation of the β subunit of xENaC in A6 cells. In vitro translated β subunit, but not α or γ, serves as a substrate for carboxylmethylation. Carboxylmethylation of ENaC reconstituted in planar lipid bilayers leads to an increase in open probability only when β subunit is present. When the channel complex is immunoprecipitated from A6 cells and analyzed by Western blot with antibodies to the three subunits of xENaC, all three subunits are recognized as constituents of the complex. The results suggest that Na+ channel activity in A6 cells is regulated, in part, by carboxylmethylation of the β subunit of xENaC.

Regulation of Epithelial Na+ Channels by Actin in Planar Lipid Bilayers and in the Xenopus Oocyte Expression System

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na+ channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na+ versus K+ permselectivity, and a substantial increase in mean open and closed times of wild-type αβγ-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the α subunit was deleted (αR613 X βγ-rENaC). 2) When αR613 X βγ-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either αR613 X βγ-rENaC or wild-type αβγ-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that α-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

Differentiation of Epithelial Na+ Channel Function AN IN VITRO MODEL

Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nm dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La3+-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (Rt) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na+ channel γ-subunit (γ-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of α- and β-ENaC and of α1- and β1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation.

Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block.

A family of novel epithelial Na+ channels (ENaCs) have recently been cloned from several different tissues. Three homologous subunits (alpha, beta, gamma-ENaCs) from the core conductive unit of Na(+)-selective, amiloride-sensitive channels that are found in epithelia. We here report the results of a study assessing the regulation of alpha,beta,gamma-rENaC by Ca2+ in planar lipid bilayers. Buffering of the bilayer bathing solutions to [Ca2+] < 1 nM increased single-channel open probability by fivefold. Further investigation of this phenomenon revealed that Ca2+ ions produced a voltage-dependent block, affecting open probability but not the unitary conductance of ENaC. Imposing a hydrostatic pressure gradient across bilayers containing alpha,beta,gamma-rENaC markedly reduced the sensitivity of these channels to inhibition by [Ca2+]. Conversely, in the nominal absence of Ca2+, the channels lost their sensitivity to mechanical stimulation. These results suggest that the previously observed mechanical activation of ENaCs reflects a release of the channels from block by Ca2+.

Point mutations in alpha bENaC regulate channel gating, ion selectivity, and sensitivity to amiloride

We have generated two site-directed mutants, K504E and K515E, in the alpha subunit of an amiloride-sensitive bovine epithelial Na+ channel, alpha bENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E alpha bENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (Ki) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E alpha bENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as being important to the regulation of fundamental channel properties.

Cation Permeability of a Cloned Rat Epithelial Amiloride‐Sensitive Na+ Channel

1 Conductance of heterotrimeric rat epithelial Na+ channels (α,β,γ‐rENaCs) for Li+ and Na+ in planar lipid bilayers was a non‐linear function of ion concentration, with a maximum of 30.4 ± 2.9 pS and 18.5 ± 1.9 pS at 1 M Li+ and Na+, respectively. 2 The α,β,γ‐rENaC conductance measured in symmetrical mixtures of Na+–Li+ (1 M) exhibited an anomalous mole fraction dependence, with a minimum at 4:1 Li+ to Na+ molar ratio. 3 Permeability ratios PK/PNa and PLi/PNa of the channel calculated from the biionic reversal potentials were dependent on ion concentration: PK/PNa was 0.11 ± 0.01, and PLi/PNa was 1.6 ± 0.3 at 50 mm; PK/PNa was 0.04 ± 0.01 and PLi/PNa was 2.5 ± 0.4 at 3 M, but differed from the ratios of single‐channel conductances in symmetrical Li+, Na+ or K+ solutions. The permeability sequence determined by either method was Li+ > Na+ > K+≫ Rb+ > Cs+. 4 Predictions of a model featuring two binding sites and three energy barriers (2S3B), and allowing double occupancy, developed on the basis of single ion current–voltage relationships, are in agreement with the observed conductance maximum in single ion experiments, conductance minimum in the mole fraction experiments, non‐linearity of the current–voltage curves in biionic experiments, and the concentration dependence of permeability ratios. 5 Computer simulations using the 2S3B model recreate the ion concentration dependencies of single‐channel conductance observed for the immunopurified bovine renal amiloride‐sensitive Na+ channel, and short‐circuit current in frog skin, thus supporting the hypothesis that ENaCs form a core conduction unit of epithelial Na+ channels.

Halothane Directly Modifies Na+ and K+ Channel Activities in Cultured Human Alveolar Epithelial Cells

During inhalational anesthesia, halogenated gases are in direct contact with the alveolar epithelium, in which they may affect transepithelial ion and fluid transport. The effects of halogenated gases in vivo on epithelial Na+ and K+ channels, which participate in alveolar liquid clearance, remain unclear. In the present study, the effects of halothane (1, 2, and 4% atm) on ion-channel function in cultured human alveolar cells were investigated using the patch-clamp technique. After exposure to 4% halothane, amiloride-sensitive whole-cell inward currents increased by 84 ± 22%, whereas tetraethylammonium-sensitive outward currents decreased by 63 ± 7%. These effects, which occurred within 30 s, remained for 30-min periods of exposure to the gas, were concentration-dependent, and were reversible upon washout. Pretreatment with amiloride prevented 90 ± 7% of the increase in inward currents without change in outward currents, consistent with an activation of amiloride-sensitive epithelial sodium channels. Tetraethylammonium obliterated 90 ± 9% of the effect of halothane on outward currents, without change in inward currents, indicating inhibition of Ca2+-activated K+ channels. These channels were identified in excised patches to be small-conductance Ca2+-activated K+ channels. These effects of halothane were not modified after the inhibition of cytosolic phospholipase A2 by aristolochic acid. Exposure of the cells to either trypsin or to low Na+ completely prevented the increase in amiloride-sensitive currents induced by halothane, suggesting a release of Na+ channels self-inhibition. Thus, halothane modifies differentially and independently Na+ and K+ permeabilities in human alveolar cells.

Opposing Effects of Bone Morphogenetic Protein-2 and Endothelin-1 on Lung Fibroblast Chloride Currents

Alteration in the control of bone morphogenetic protein (BMP)-regulated genes and increased expression of endothelin (ET)-1 are both believed to play important roles in the still incompletely understood pathobiology of pulmonary vascular remodeling and fibrosis. Recent studies have drawn attention to the contribution of adventitial fibroblast activation in these phenomena. Because chloride channels are involved in the control of physiological function of fibroblasts, we hypothesized that these channels are differentially regulated by BMPs and ET. We measured chloride ion currents by whole-cell path-clamping in cultured primary human pulmonary fibroblasts. The application of BMP2 prevented activation of these currents by hypotonic challenge in a time- and dose-dependent manner, partially via protein kinase C signaling. Maximal inhibition was observed after 45-minute incubation of cells in the presence of 10 ng/ml of BMP2. ET-1 did not activate chloride channels acutely; however, prolonged treatment of cells with ET-1 (100 nM, 2 h) induced the appearance of lysophosphatidic acid-activated chloride currents (a marker of differentiated myofibroblasts), and this induction could be effectively blocked by BMP2 pretreatment (10 ng/ml). BMP2 also prevented stimulation of α-smooth muscle actin gene expression and cell migration of fibroblasts induced by ET-1. We conclude that ET-1 and BMP2 have opposing effects on chloride channel activity in human fibroblasts. This is a potentially relevant mechanism involved in pulmonary vascular remodeling and fibrosis.

Electrophysiological Characterization of Rat Type II Pneumocytes in Situ

Optimal aeration of the lungs is dependent on an alveolar fluid clearance, a process that is governed by Na+ and Cl- transport. However, the specific contribution of various ion channels in different alveolar cell types under basal or stimulated conditions is not exactly known. We established a novel functional model of rat lung slices suitable for nystatin-perforated whole-cell patch-clamp experiments. Lung slices retained a majority of live cells for up to 72 hours. Type II pneumocytes in situ had a mean capacitance of 8.8 +/- 2.5 pF and a resting membrane potential of -4.4 +/- 1.9 mV. Bath replacement of Na+ with NMDG+ decreased inward whole-cell currents by 70%, 21% and 52% of which were sensitive to 10 microM and 1 mM of amiloride, respectively. Exposure of slices to 0.5 microM dexamethasone for 1 hour did not affect ion currents, while chronic exposure (0.5 microM, 24-72 h) induced an increase in both total Na+-entry currents and amiloride-sensitive currents. Under acute exposure to 100 microM cpt-cAMP, Type II cells in situ rapidly hyperpolarized by 25-30 mV, due to activation of whole-cell Cl- currents sensitive to 0.1 mM of 5-Nitro-2-(3-phenylpropylamino)benzoic acid. In addition, in the presence of cpt-cAMP, total sodium currents and currents sensitive to 10 microM amiloride increased by 32% and 70%, respectively. Thus, in Type II pneumocytes in situ: (1) amiloride-sensitive sodium channels contribute to only half of total Na+-entry and are stimulated by chronic exposure to glucocorticoids; (2) acute increase in cellular cAMP content simultaneously stimulates the entry of Cl- and Na+ ions.