Apical Anion Conductance Research Articles

Discovering the chloride pathway in the CFTR channel

Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure–function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.

The distribution and expression of CFTR restricts electrogenic anion secretion to the ileum of the brushtail possum, Trichosurus vulpecula

In eutherian mammals, fluid secretion is essential for intestinal function. This is driven by electrogenic Cl(-) secretion, which involves a NaK2Cl cotransporter (NKCC1) in the enterocyte basolateral membrane and the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, in the possum ileum, NKCC1 expression is low and secretagogues stimulate electrogenic HCO(3)(-) secretion driven by a basolateral NaHCO(3) cotransporter (pNBCe1). Here we investigated whether electrogenic anion secretion occurs in possum duodenum and jejunum and determined the role of CFTR in possum intestinal anion secretion. Prostaglandin E(2) (PGE(2)) and forskolin stimulated a large increase in ileal short-circuit current (I(sc)), consistent with electrogenic HCO(3)(-) secretion, but had little effect on the duodenal and jejunal I(sc). Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH101) inhibited cloned possum CFTR in cultured cells and the PGE(2)-stimulated ileal I(sc), implicating CFTR in ileal HCO(3)(-) secretion. Consistent with this, CFTR is expressed in the apical membrane of ileal crypt and lower villous cells, which also express pNBCe1 in the basolateral membrane. In contrast, duodenal and jejunal CFTR expression is low relative to the ileum. Jejunal pNBCe1 expression is also low, whereas duodenal and ileal pNBCe1 expression are comparable. All regions have low NKCC1 expression. These results indicate that cAMP-dependent electrogenic Cl(-) secretion does not occur in the possum small intestine because of the absence of CFTR and NKCC1. Furthermore, CFTR functions as the apical anion conductance associated with HCO(3)(-) secretion and its distribution limits electrogenic HCO(3)(-) secretion to the ileum.

Paradoxical Effects of Hydrogen Peroxide on Human Airway Anion Secretion

The present study concerns intriguing effects of hydrogen peroxide (H2O2) on cAMP-mediated anion secretion in polarized human airway epithelia. Although H2O2 applied to the apical and basolateral membrane increases short-circuit currents (ISC) with analogous properties, it has opposite effects on subsequent cAMP-activated ISC responses. Namely, forskolin (FK)-induced ISC responses were down-regulated by the apical presence of H2O2, whereas they were up-regulated by its basolateral presence. Despite this contrasting effect, oxidative stimuli from either aspect of the monolayer hindered FK-induced increments in cytosolic cAMP levels and apical membrane Cl- conductance. The site-dependent effects of H2O2 were reproduced in the responses to 8-bromo-cAMP. Estimation of the anionic composition of the ISC revealed that the FK up-regulated both bumetanide [an Na+-K+-2Cl- cotransporter (NKCC1) inhibitor]-sensitive and 4,4'-dinitrostilbene-2,2'-disulfonic acid [an HCO3--dependent anion transporter (NBC1/AE2) inhibitor]-sensitive ISC in the control, whereas the up-regulation evidently favored bumetanide-sensitive ISC in the basolateral presence of H2O2. The FK-induced NKCC1 augmentation after exposure to basolateral H2O2 was counteracted by cytochalasin D, an inhibitor of microfilament function, but not by charybdotoxin, a blocker of the intermediate conductance Ca2+-activated K+ channel, whose activation could be related to NKCC1-mediated Cl- secretion. These observations suggest that basolaterally but not apically applied H2O2 potentiates subsequent cAMP-mediated Cl- secretion by an increase in Cl- uptake via basolateral NKCC1, whose sensitivities to cAMP/protein kinase A are up-regulated, overcoming the H2O2-induced inhibition of cAMP-mediated apical anion conductance. The basolateral membrane-specific effects of H2O2 may be relevant to the basolateral cytoskeleton, which is believed to interact with NKCC1.

Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells.

Interleukin (IL)-13 has been associated with asthma, allergic rhinitis, and chronic sinusitis, all conditions where an imbalance in epithelial fluid secretion and absorption could impact upon the disease. We have investigated the effects of IL-13 on the ion transport characteristics of human bronchial epithelial cells cultured at an apical-air interface. Ussing chamber studies indicated that 48 h pretreatment with IL-13 or IL-4 significantly reduced the basal short-circuit current (I(sc)) and inhibited the amiloride-sensitive current by >98%. Furthermore, the I(sc) responses were increased by more than six- and twofold over control values when stimulated with UTP or forskolin, respectively, after cytokine treatment. The IL-13-enhanced response to UTP/ionomycin was sensitive to bumetanide and DIDS and was reduced in a low-chloride, bicarbonate-free solution. Membrane permeablization studies indicated that IL-13 induced the functional expression of an apical Ca(2+)-activated anion conductance and that changes in apical or basolateral K(+) conductances could not account for the increased I(sc) responses to UTP or ionomycin. The results indicate that IL-13 converts the human bronchial epithelium from an absorptive to a secretory phenotype that is the result of loss of amiloride-sensitive current and an increase in a DIDS-sensitive apical anion conductance.

Diphenylamine-2-carboxylic acid (DPC), Usually an inhibitor of Cl- and non-selective cation channels, inhibits Cl-/HCO3- exchange and opens Cl- and cation conductances in rabbit gallbladder epithelium.

In the apical plasma membrane of rabbit gallbladder epithelium various drugs (hydrochlorothiazide, phlorizin, phenylglyoxal) inhibit Cl-/HCO3- exchange and probably enhance the almost negligible intrinsic anion conductance of the exchanger. By radiochemical measurements of apical Cl- influx, the anion exchange is shown here to be directly and immediately inhibited by diphenylamine-2-carboxylic acid (DPC) too. Using conventional microelectrode techniques in intact tissue, DPC, with same dose/response curve, is shown to activate an apical anion conductance (GCl) that has similar properties and amplitude to the GCl activated by the other exchange inhibitors so far tested; the actions are not additive. Patch-clamp methods (cell-attached and excised inside-out patch configurations) reveal that GCl is due to anion channels that are non-rectifying, cytoplasm independent, sensitive to stilbene and dipyridamole and have conductance of a few picosiemens. All this strengthens the correlation between inhibition of anion exchange and the activation of GCl and channels with features similar to those of the almost negligible intrinsic anion conductance of the exchanger. Among the drugs tested, the effects of DPC and hydrochlorothiazide are even more similar, such that even their dose/response curves overlap. Moreover, both drugs also directly activate some verapamil-sensitive Ca2+ channels and consequently apamin-sensitive, Ca2+-activated K+ channels. Thus DPC, usually an inhibitor of Cl- and non-selective cation channels, is shown here to be capable of activating Cl- and cation conductances.

Cryptdin-3 induces novel apical conductance(s) in Cl- secretory, including cystic fibrosis, epithelia.

Opening of anion-conductive pathways in apical membranes of secretory cells lining mucosal surfaces is a critical step in salt and water secretion and, thus, hydration of sites including airway and intestine. In intestine, Paneth cells are positioned at the base of the secretory gland (crypt) and release defensin peptide, in mice termed cryptdins, into the crypt lumen. Because at least some defensins have been shown to form anion-conductive channels in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes. To directly evaluate the possibility of cryptdin-3-mediated apical anion conductance (G(ap)), we have utilized amphotericin B to selectively permeabilize basolateral membranes of electrically tight monolayers of polarized human intestinal secretory epithelia (T84 cells), thus isolating the apical membrane for study. Cryptdin-3 induces G(ap) that is voltage independent (deltaG(ap) = 1.90 +/- 0.60 mS/cm2) and exhibits ion selectivity contrasting to that elicited by forskolin or thapsigargin (for cryptdin-3, Cl- = gluconate; for forskolin and thapsigargin, Cl- >> gluconate). We cannot exclude the possibility that the macroscopic current induced by cryptdin could be the sum of cation and Cl- currents. Cryptdin-3 induces a current in basolaterally permeabilized epithelial monolayers derived from airway cells harboring the deltaF508 mutation of cystic fibrosis (CF; deltaG(ap) = 0.80 +/- 0.06 mS/cm2), demonstrating that cryptdin-3 restores anion secretion in CF cells; this occurs independently of the CF transmembrane conductance regulator channel. These results support the idea that cryptdin-3 may associate with apical membranes of Cl--secreting epithelia and self-assemble into conducting channels capable of mediating a physiological response.

Inhibitors of the Cl-/HCO3- exchanger activate an apical anion conductance with similar features in the epithelial cells of rabbit gallbladder: analysis in intact epithelium.

In the apical plasma membrane of rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ), besides its inhibiting action on Na+-Cl- symport and some side-effects, opens a SITS-sensitive Cl- conductance (Gcl), resulting in depolarization of the membrane due to a cell-to-lumen Cl- backflux. Some previous indications suggest that GCl is someway related to the Cl-/HCO3- exchanger. Thus the actions on the apical membrane of HCTZ and two inhibitors of the exchanger mainly studied in erythrocytes, namely phlorizin and phenylglyoxal (PG), have been compared. The transapical Cl- influx was measured radiochemically and the anion exchange fraction identified. The apical and transepithelial membrane potentials (Vm, Vm), the apical/basolateral membrane resistance ratio (Rm/Rs) and the transepithelial resistance (Rep) were measured with conventional microelectrodes and the changes in apical electromotive force and conductance were calculated. It has been shown that: (1) 2.5 x 10(-4) mol/l HCTZ abolishes Cl-/HCO3- exchange in a few seconds, (2) exchanger inhibition by HCTZ is direct and not mediated by carbonic anhydrase inhibition, (3) 2 mmol/l phlorizin and 4 mmol/l PG also inhibit the exchanger in a few seconds or at least rapidly and in parallel activate a depolarization of 6-7 mV, like HCTZ, (4) dose/response curves of the three drugs for depolarization activation and anion exchange inhibition overlap, (5) depolarization time courses are similar for the three drugs, (6) a decrease in the Rm/Rs ratio occurs in the presence of the three drugs, with a significant change in apical electromotive force when the luminal Cl- concentration is reduced, all this indicating the appearance of a substantial, quantifiable GCl which is absent in the absence of incubation with the drugs, (7) GCl values are similar, regardless of the drug which generates them, (8) the effects of the three drugs are not additive, and (9) stilbenes and dipyridamole abolish GCl (but not DPC) as well as the basal intrinsic conductance of the exchanger. On this basis it is concluded that some inhibitors of the Cl-/HCO3- exchanger either turn it into an anion channel or, less probably, activate a parallel GCl, as a consequence of the exchanger inhibition.

Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium.

Anion selectivity of the cystic fibrosis conductance transmembrane conductance regulator (CFTR) and other channels and parallel pathways expressed endogenously in apical membranes of polarized Calu-3 epithelial monolayers was studied under control conditions and during cAMP stimulation. Basolateral membranes were eliminated using alpha-toxin. The cAMP-stimulated, gradient-driven currents had the sequence Br>/=Cl>/=NO3>SCN> I>/=F>formate>HCO3>acetate>propionate=butyrate=ATP= PPi=PO4=SO4=0. Selectivity of parallel cAMP-independent pathway(s) was Br>Cl=SCN=NO3>I>formate=F >HCO3>acetate>propionate. SCN, I, F or formate blocked cAMP-stimulated, but not control, Cl currents. Anions >0.53 nm in diameter were impermeant, suggesting that the apical CFTR channel has a limiting diameter of 0.53 nm. The selectivity, blocking patterns and pore size of the cAMP-stimulated conductance pathway were very similar to those in previous reports in which CFTR was heterologously expressed in non-epithelial cells. Thus, CFTR appears to be the major apical anion conductance pathway in Calu-3 cells, and its conduction properties are independent of the expression system. CFTR in Calu-3 cells also conducts physiologically relevant anions, but not ATP, PO4 or SO4. A pathway parallel (probably a tight junction) showed a different selectivity than CFTR.

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

Studies of transepithelial Cl- transport in cultured cauda epididymal cells of rats by the short-circuit current method.

1. Monolayer cultures of cauda epididymides from male Sprague-Dawley rats (210-230 g) were studied by the short-circuit current (ISC) technique to characterize the properties of the transepithelial chloride transport. In HCO(3-)-free, HEPES-buffered solution, adrenaline (0.23 microM) added to the basolateral side led to an increase in ISC and transepithelial conductance (gt). 2. Decreasing apical chloride concentration ([Cl-]a) progressively from 126.7 to 0 mM by substituting chloride with gluconate increased the ISC response to adrenaline (delta ISC) in a linear fashion with a slope of -1.6 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. Pretreating the tissue with a chloride channel blocker diphenylamine-2-carboxylate (DPC) on the apical side significantly reduced the slope to -4.9 x 10(-4) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. 3. By substituting apical chloride with various anions and measuring the change in ISC upon adrenaline stimulation, the selectivity sequence of the apical anion conductance was found to be NO3- approximately Br- > Cl- > I- > gluconate > isethionate. 4. When the monolayers were bathed with Krebs-Henseleit solution containing 25 mM HCO3- and 5% CO2, the delta ISC at each [Cl-]a as well as the dependence of delta ISC on [Cl-]a (slope = -3.3 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a) were significantly greater than the HCO(3-)-free counterpart. Addition of 0.1 mM acetazolamide or 0.5 mM SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) to the basolateral side significantly reduced the effects of HCO3- and CO2. 5. When the tissues were bathed on both sides with HCO(3-)-free, HEPES-buffered solution and were clamped at various transepithelial potential differences (PDt) from +30 mV (lumen positive) to -30 mV (lumen negative), the relationship between the clamping current response to adrenaline (delta ICL) and the PDt applied was linear. Zero clamping current response was found at -6 mV. Decreasing [Cl-]a to 0 mM reduced the dependence of delta ICL on PDt and delta ICL was positive at all PDt tested. The response of the transepithelial conductance to adrenaline (delta gt) did not depend on the PDt applied but was reduced with decreasing apical chloride concentration.(ABSTRACT TRUNCATED AT 400 WORDS)