Cystic fibrosis transmembrane conductance regulator chloride channel lockers: Pharmacological,biophysical and physiological relevance Linsdell P简

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR channel structure and function, and of how CFTR activity is controlled by its local environment.     

Cystic fibrosis transmembrane conductance regulator chloride channel blockers:Pharmacological,biophysical and physiological relevance

Dysfunction of the cystic fibrosis transmembrane con-ductance regulator(CFTR) chloride channel causes cys-tic fibrosis, while inappropriate activity of this channeloccurs in secretory diarrhea and polycystic kidney dis-ease. Drugs that interact directly with CFTR are there-fore of interest in the treatment of a number of diseasestates. This review focuses on one class of small mol-ecules that interacts directly with CFTR, namely inhibi-tors that act by directly blocking chloride movementthrough the open channel pore. In theory such com-pounds could be of use in the treatment of diarrheaand polycystic kidney disease, however in practice allknown substances acting by this mechanism to inhibitCFTR function lack either the potency or specificity forin vivo use. Nevertheless, this theoretical pharmaco-logical usefulness set the scene for the developmentof more potent, specific CFTR inhibitors. Biophysically,open channel blockers have proven most useful as ex-perimental probes of the structure and function of theCFTR chloride channel pore. Most importantly, the useof these blockers has been fundamental in developing afunctional model of the pore that includes a wide innervestibule that uses positively charged amino acid sidechains to attract both permeant and blocking anionsfrom the cell cytoplasm. CFTR channels are also subjectto this kind of blocking action by endogenous anionspresent in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physio-logical control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR chan-nel structure and function, and of how CFTR activity is controlled by its local environment.     

Cellular mechanism of adrenalin stimulated chloride secretion via beta-adrenoceptor in T84 cells

In the present study, the intracellular regulatory pathways involved in the adrenalin-stimulated chloride secretion across T84 cells were investigated. Biphasic characteristics were observed in the Isc response to the basolateral addition of adrenalin (0.25 nM-100 microM). The biphasic response was almost abolished by removing ambient Cl(-). Chloride secretion was found to depend on the activities of basolaterally located Na+-K+-2Cl(-) cotransporters and K+ channels. The alpha-adrenoceptor antagonist phentolamine did not have any effect on either phase of adrenalin-induced Isc, while after pretreatment of the beta-adrenoceptor antagonist propranolol, the adrenalin-induced Isc was substantially abolished, suggesting the biphasic response may be mediated by the beta-adrenoceptor. Under whole cell patch-clamp conditions, T84 cells responded to adrenalin with a rise in inward current. The current, which exhibited a linear I-V relationship and time- and voltage-independent characteristics, was inhibited by the chloride channel blocker DPC and the reverse potential was close to the equilibrium potential for Cl(-) (0 mV), implying that the current was Cl(-) selective. When preloaded with a Ca2+-chelating agent, BAPTA/AM did not affect the Isc response to adrenalin, whereas the Isc was destroyed by pretreating the cells with an adenyl cyclase inhibitor, MDL12330A. These observations were further supported by the intracellular [cAMP] measurement experiment, indicating that adrenalin induced chloride secretion could be mediated by a beta-adrenoceptor only involving cAMP as an intracellular second messenger.     

Activation of chloride conductance in pig jejunal brush border vesicles

Summary Requirements for the activation of Cl conductance have been investigated in pig jejunal brush border vesicles. The stability of ATP as a substrate for protein kinase activity, the stability of the phosphoprotein product of protein kinase action, and the choice of buffer system used for vesicle preparation were studied as variables which affected the outcome of in vitro activation attempts. Arsenate was selected as the most effective agent in protecting ATP from hydrolysis by the phosphatase activity in this vesicle system. Brush border vesicle protein appeared to prevent the accumulation of phosphoprotein in a cAMP-dependent protein kinase reaction, and vesicle protein only had phosphate acceptor activity when KF was added as a presumptive inhibitor of phosphoprotein phosphatase.A Cl conductance response to a potassium gradient and valinomycin was present in vesicles prepared in buffers containing tetramethylammonium. Cl^– conductance activity was not increased in this system by the addition of ATP, dibutyryl cyclic AMP, and cyclic AMP-dependent protein kinase.There was no Cl conductance response to a potassium gradient in vesicles buffered with imidazolium-acetate. Incorporation of ATP, AsO ₄ ^3– , and F^– into these nonconductive vesicles by homogenization, followed by addition of dibutyryl cAMP, produced substantial conductance activity. Maximal activation of Cl^– conductance was obtained with vesicles prepared in imidazolium-acetate buffering, using precautions to stabilize ATP and phosphoprotein prior to conductance measurements.     

Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.     

Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore

Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^? channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl^? channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.     

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ～110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 μM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of ～1 μM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.     

Effects of mutations in cAMP-dependent protein kinase on chloride efflux in Caco-2 human colonic carcinoma cells

In order to evaluate the importance of cAMP and cAMP-dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco-2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulator subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn²⁺-inducible manner and exhibited Zn²⁺-inducible inhibition of cAMPdPK activity. The parental and transformed Caco-2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide-efflux assay. In the transformants, induction of the protein kinase mutation with ZnSo₄ markedly decreased chloride efflux in response to forskolin, the 8-(4-chlorophenylthio) analog of cAMP, vasoactive intestial polypeptide, prostaglandin E2 and isoproterenol, whereas Zn²⁺-treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue-regulated chloride channel activity in the Caco-2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase-defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation. © 1995 Wiley-Liss, Inc.     

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl^? and HCO₃^? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO₃^? efflux through CFTR often required the intracellular H⁺/HCO₃^? production and/or the Na⁺-dependent basolateral HCO₃^? uptake, the intracellular pH (pH_i) balance might be disturbed, especially as a consequence of increased endogenous H⁺ and HCO₃^? production. However, measurement of pH_i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO₃^? transport. In addition, although the plasma membrane Na⁺/K⁺-ATPase (NKA) is normally essential for basolateral HCO₃^? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH_i or NKA abundance on cell membrane.     

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl⁻ and could be reversibly blocked by the Cl⁻ channel blockers 4-acetamido-4′-isothio-cyanatistilbene-2,2′disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl⁻]_i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl⁻ conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K⁺ or Na⁺ ruled out the possibility that the Cl⁻ conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca²⁺ imaging experiments showed that betaine did not affect [Ca²⁺]_i, suggesting that the Cl⁻ current is not Ca²⁺ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons. Accepted: 20 March 1998     

Activation of γ-aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin B1a

The interaction of avermectin B_1a (AVMB_1a) with mouse brain chloride channels was characterized using a radiochloride efflux assay. The loss of intravesicular chloride from synaptoneurosomes preloaded with ³⁶Cl involved an initial rapid phase followed by a slower phase that approached equilibrium within 10 min. AVMB_1a stimulated a 30% loss of intravesicular chloride within the first 2 s of exposure; however, AVMB_1a had no effect on the rate of the slower phase of chloride loss. Experiments with lysed synaptoneurosomes showed that both chloride loading and basal and AVMB_1a-stimulated chloride release required the presence of intact vesicles. The efflux of ³⁶Cl from mouse brain synaptosomes and the stimulation of efflux by AVMB_1a were qualitatively similar to the results obtained with synaptoneurosomes but involved much lower overall levels of chloride loading and release. AVMB_1a produced halfmaximal stimulation of chloride efflux from synaptoneurosomes at a concentration of 2.1 ± 0.3 μM and a 35.4 ± 1.4% maximal loss of intravesicular chloride at saturating concentrations. γ-Aminobutyric acid (GABA), bicuculline, or the chloride channel blockers picrotoxinin, t-butylbicyclophosphorothionate (TBPS) 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and anthracene 9-carboxylic acid (9-CA) had little or no effect on the loss of chloride from synaptoneurosomes either in the presence or the absence of AVMB_1a. However, the chlorinated cycloalkane insecticides dieldrin and lindane were equally effective as inhibitors of GABA-dependent chloride uptake and AVMB_1a-stimulated chloride efflux. These data demonstrate that AVMB_1a-stimulated chloride efflux from mouse brain synaptic vesicles results from the activation of GABA-insensitive chloride channels and that this action is distinct from their previously documented effects on GABA-gated chloride channels in mouse brain preparations. Our findings imply that both GABA-gated and GABA-insensitive chloride channels may be toxicologically significant targets for the action of avermectins.     

Calcium-activated chloride conductance of lactotrophs: Comparison of activation in normal and tumoral cells during thyrotropin-releasing-hormone stimulation

We studied a chloride (Cl-) conductance activated by calcium (Ca2+) in normal rat lactotrophs and compared its activation during TRH stimulation in normal rat lactotrophs and in GH3 tumoral lactosomatotrophs cells, using the whole-cell configuration of the patch-clamp technique. The Cl- specificity of the conductance was assessed by manipulation of internal and external Cl- concentrations. The reversal potentials were in agreement with those predicted by the Nernst equation. Ca2+ ionophore A23187 and membrane depolarizations activated the Cl- conductance. However, a feedback effect of Cl- gradient modifications on Ca2+ movements was also observed in normal lactotrophs. In the latter, TRH (100 nM) mobilization of intracellular Ca2+ activated this Cl- conductance together with the potassium (K+) conductance when both ions were present in the intracellular medium (IM) or alone when K+ was absent. Chloride conductance was not activated in the GH3 cells, where mobilization of intracellular Ca2+ by TRH (100 nM) activated only Ca2(+)-dependent K+ conductance. It seems likely that the activation of Cl- conductance in these two different cell types involves different mechanisms.     

Genomic, Tissue Expression, and Protein Characterization of pCLCA1, a Putative Modulator of Cystic Fibrosis in the Pig

Recent studies have identified members of the CLCA (chloride channels, calcium-activated) gene family as potential modulators of the cystic fibrosis (CF) phenotype, but differences between the human and murine CLCA genes and proteins may limit the use of murine CF models. Recently established pig models of CF are expected to mimic the human disease more closely than the available mouse models do. Here, we characterized the porcine CLCA gene locus, analyzed the expression pattern and protein processing of pCLCA1, and compared it to its human ortholog, hCLCA1. The porcine CLCA gene family is located on chromosome 4q25, with a broad synteny with the human and murine clca gene loci, except for a pig-specific gene duplication of pCLCA4. Using pCLCA1-specific antibodies, the protein was immunohistochemically localized in mucin-producing cells, including goblet cells and mucinous glands in the respiratory and alimentary tracts. Similar to hCLCA1, biochemical characterization of pCLCA1 identified a secreted soluble protein that could serve as an extracellular signaling molecule or functional constituent of the protective mucous layers. The results suggest that pCLCA1 shares essential characteristics of hCLCA1, supporting the pig model as a promising tool for studying the modulating role of pCLCA1 in the complex pathology of CF. (J Histochem Cytochem 57:1169–1181, 2009)     

Cholesterol-dependent regulation of adenosine A2A receptor-mediated anion secretion in colon epithelial cells

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. In this study we have investigated the role of cholesterol in the adenosine-dependent regulation of ion transport in colonic epithelial cells. We observed that methyl-β-cyclodextrin (MβCD), a cholesterol-sequestering molecule, enhanced adenosine A_2A receptor-activated transepithelial short circuit current (I_sc), but only from the basolateral side. Cholesterol is a major constituent of membrane microdomains, called lipid rafts that also contain sphingolipids. However, studies with the sphingomyelin-degrading enzyme, sphingomyelinase, and the cholesterol-binding agent, filipin, indicated that the change in the level of cholesterol alone was sufficient to control the adenosine-modulated I_sc. Cholesterol depletion had a major effect on the functional selectivity of A_2A receptors. Under control conditions, adenosine activated I_sc more potently than the specific A_2A agonist, CGS-21680, and the current was inhibited by XE991, an inhibitor of cAMP-dependent K⁺ channels. Following cholesterol depletion, CGS-21680 activated I_sc more potently than adenosine, and the current was inhibited by clotrimazole, an inhibitor of Ca²⁺-activated K⁺ (IK1) channels. Co-immunoprecipitation experiments revealed that A_2A receptors associate with IK1 channels following cholesterol depletion. These results suggest that cholesterol content in colonic epithelia affects adenosine-mediated anion secretion by controlling agonist-selective signaling.     

cAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

Summary The role of adenosine 3,5-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl^– conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100nm catalytic subunit of PK-A purified from human placenta and 1mm of the phosphatase resistant adenosine 5-thiotriphosphate (ATP-gamma-S) and resealed. Cl^– conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23°C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([¹⁴C]-sucrose) and included in each Cl^– flux calculation. In 19 independent experiments, the mean Cl^– conductance in placental membranes in the absence of phosphorylation was 3.67±3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97±1.75 nmol·sec^–1·(mg protein)^–1 (mean±sd). PK-A dependent phosphorylation reduced the Cl^– conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl^– channels, confirmed that the PK-A dependent phosphorylation increased the Cl^– conductance in 11/13 experiments, from 1.01±0.61 to 1.85±0.99 nmol·sec^–1·(mg protein)^–1(mean±sd). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl^– channel(s) of the apical membranes of human placenta.     

Asymmetry in the Osmotic Response of a Rat Cortical Collecting Duct Cell Line: Role of Aquaporin-2

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl⁻) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl⁻ current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl⁻ current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G_j). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.     

Aquaporin-4 as a molecular partner of cystic fibrosis transmembrane conductance regulator in rat Sertoli cells

Sertoli cells (SCs) form the blood–testis barrier (BTB) that controls the microenvironment where the germ cells develop. The cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role to male fertility and it was recently suggested that it may promote water transport. Interestingly, Aquaporin-4 (AQP4) is widely expressed in blood barriers, but was never identified in SCs. Herein we hypothesized that SCs express CFTR and AQP4 and that they can physically interact.