Small molecule anionophores promote transmembrane anion permeation matching CFTR activity

Anion selective ionophores, anionophores, are small molecules capable of facilitating the transmembrane transport of anions. Inspired in the structure of natural product prodigiosin, four novel anionophores 1a-d, including a 1,2,3-triazole group, were prepared. These compounds proved highly efficient anion exchangers in model phospholipid liposomes. The changes in the hydrogen bond cleft modified the anion transport selectivity exhibited by these compounds compared to prodigiosin and suppressed the characteristic high toxicity of the natural product. Their activity as anionophores in living cells was studied and chloride efflux and iodine influx from living cells mediated by these derivatives was demonstrated. These compounds were shown to permeabilize cellular membranes to halides with efficiencies close to the natural anion channel CFTR at doses that do not compromise cellular viability. Remarkably, optimal transport efficiency was measured in the presence of pH gradients mimicking those found in the airway epithelia of Cystic Fibrosis patients. These results support the viability of developing small molecule anionophores as anion channel protein surrogates with potential applications in the treatment of conditions such as Cystic Fibrosis derived from the malfunction of natural anion transport mechanisms.European Union’s Horizon 2020 research and innovation programme under grant agreement No. 667079, La Marató de TV3 Foundation (20132730), Consejería de Educación de la Junta de Castilla y León (Projects BU340U13 and BU092U16

High-Throughput Screening for Modulators of CFTR Activity Based on Genetically Engineered Cystic Fibrosis Disease-Specific iPSCs

Organotypic culture systems from disease-specific induced pluripotent stem cells (iPSCs) exhibit obvious advantages compared with immortalized cell lines and primary cell cultures, but implementation of iPSC-based high-throughput (HT) assays is still technically challenging. Here, we demonstrate the development and conduction of an organotypic HT Cl/I exchange assay using cystic fibrosis (CF) disease-specific iPSCs. The introduction of a halide-sensitive YFP variant enabled automated quantitative measurement of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in iPSC-derived intestinal epithelia. CFTR function was partially rescued by treatment with VX-770 and VX-809, and seamless gene correction of the p.Phe508del mutation resulted in full restoration of CFTR function. The identification of a series of validated primary hits that improve the function of p.Phe508del CFTR from a library of 42,500 chemical compounds demonstrates that the advantages of complex iPSC-derived culture systems for disease modeling can also be utilized for drug screening in a true HT format

Efficient, non‐toxic anion transport by synthetic carriers in cells and epithelia

Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.info:eu-repo/semantics/publishe

IONIC SELECTIVITY OF VOLUME-SENSITIVE CURRENTS IN HUMAN EPITHELIAL-CELLS

The ion selectivity of swelling-activated Cl- currents has been investigated in three different human epithelial cell lines, two derived from the airway epithelium (9HTEo- and CFNPE9o-) and one from a colon carcinoma (T84). The relative permeability of volume-sensitive currents with respect to Cl- is: I- (1.19) greater than NO3- (1.07) approximately Br-(1.05) greater than Cl-(1.0) greater than F-(0.5) approximately HCO3-(0.48) greater than isethionate(0.28) greater than aspartate (0.14) approximately gluconate(0.13) approximately SO4(2-)(0.12). This type of ion selectivity is similar to that described for depolarization-activated outwardly rectifying Cl- channels found in epithelial cells

A FORSKOLIN AND VERAPAMIL SENSITIVE K+ CURRENT IN HUMAN TRACHEAL CELLS

A voltage-dependent K+ current has been revealed in whole-cell recordings carried out on immortalized cells obtained from the human tracheal epithelium. At positive membrane potentials the current shows a time dependent inactivation which is accelerated by increasing the depolarizing step. Forskolin, a direct activator of adenylyl cyclase, and verapamil, a Ca2+ channel blocker, induce the K+ current to inactivate more rapidly. Control experiments show that the action of these two compounds is not mediated by cyclic AMP and Ca2+. The application of 1,9-dideoxyforskolin, an analogue which does not stimulate adenylate cyclase, inhibits the current in the same way as forskolin; on the contrary, the dibutyryl analogue of cyclic AMP is ineffective. Furthermore, eliminating extracellular Ca2+ does not affect K+ current kinetics. Tetraethylammonium is an effective blocker of this current with an IC50 of 0.3 mM

HIV-1 Nef expression inhibits the activity of a Ca2+-dependent K+ channel involved in the control of the resting potential in CEM lymphocytes

The HIV-1 Nef protein plays an important role in the development of the pathology associated with AIDS. Despite various studies that have dealt with different aspects of Nef function, the complete mechanism by which it alters the physiology of infected cells remains to be established. Nef can associate with cell membranes, therefore supporting the hypothesis that it might interact with membrane proteins as ionic channels and modify their electrical properties. By using the patch-clamp technique, we found that Nef expression determines a 25-mV depolarization of lymphoblastoid CEM cells. Both charybdotoxin (CTX) and the membrane-permeable Ca2+ chelator BAPTA/AM depolarized the membrane of native cells without modifying that of Nef-transfected cells. These data suggested that the resting potential in native CEM cells is settled by a CTX- and Ca2+-sensitive K+ channel (KCa,CTX), whose activity is absent in Nef-expressing cells. This was confirmed by direct measurements of whole-cell KCa,CTX currents. Single-channel recordings on excised patches showed that a KCa,CTX channel of 35 pS with a half-activation near 400 nM Ca2+ was present in both native and Nef-transfected cells. The measurements of free intracellular Ca2+ were not different in the two cell lines, but Nef-transfected cells displayed an increased Ca2+ content in ionomycin-sensitive stores. Taken together, these results indicate that Nef expression alters the resting membrane potential of the T lymphocyte cell line by inhibiting a KCa,CTX channel, possibly by intervening in the regulation of intracellular Ca2+ homeostasis

EXTRACELLULAR 2-CHLOROADENOSINE AND ATP STIMULATE VOLUME- SENSITIVE CL- CURRENT AND CALCIUM MOBILIZATION IN HUMAN TRACHEAL 9HTEO- CELLS

The perforated-patch whole-cell technique was used to record membrane currents in epithelial cells (9HTEo-) obtained from the human tracheal epithelium. Extracellular application of 2-chloroadenosine and ATP (0.01-100 microM) caused activation of Cl- currents similar to those regulated by cell volume in airway and intestinal cells. This response was inhibited by increasing extracellular osmolality, by omission of extracellular Ca2+, or by the addition of the A2 adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Fluorimetric measurements with fura-2 reveal that 2-chloroadenosine and ATP elicited both a Ca2+ influx through the plasma membrane and a release from intracellular stores

Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of th

We need better values

In many developing countries there has been, and still is, a strong move for the government to give up or reduce its involvement in ventures that the market can take care of. The ideological basis for this hinges on the belief that the government should not provide goods and services that the private sector is willing and able to provide. Rather, the government should be concerned with providing goods and services that are public in nature but have the potential for market failure. This has been behind the moves for privatisation of veterinary services all over the world. In Ghana, the government has been responsible for the provision of veterinary services to animal owners freely or at subsidised levels since independence. However, there have been arguments against continued free provision or subsidy because the government is facing budgetary and financial difficulties. Another argument is that over-employment is over-extending the ability of government to provide quality services, and that some of those employed by the government have to go into the private sector to ease the burden. A project to help privatise veterinary services was launched in 1993, encouraging veterinarians to go into private practice. However, one factor critical to the success of the privatisation effort is the willingness of livestock farmers or animal owners to pay the full cost for animal health services. Their willingness to pay is to a large extent influenced by the livestock market structure, since services have to be paid for with cash in most cases. There is, though, no well-defined livestock marketing structure in Ghana. Livestock producers and consumers are therefore at the mercy of middlemen whose aim is to maximise profits by buying animals cheaply and selling meat at high prices to consumers. No formal market structures exist at present. Sometime back, the government set up a Meat Marketing Board that was charged with buying livestock from farmers and processing the meat to sell at reasonably competitive prices. However, the Board is now defunct partly because of low farmer patronage. One reason was that farmers were not paid on time and payments were on a carcass weight basis, which was not attractive. The alternative was for livestock owners to sell on the hoof to middlemen for ready cash. Middlemen too dominant The middlemen they are mainly butchers and influential people from livestock producing communities have a monopoly and therefore dictate what to pay for animals and what the price of meat is. Livestock farmers are not well organised, and therefore lack strong bargaining power to deal with the hegemony of the middlemen. If the prevailing market structure is left to stand, livestock farmers will continue to receive low prices for their animals and will therefore not have the financial power to pay for services rendered by private veterinarians. The effect is that private practice will not be profitable and will thus discourage other veterinarians from going in. Another factor in the market structure with implications for privatisation of services is the value system of livestock owners. Farmers are more concerned about numbers than quality. They are reluctant to sell animals. Animals are therefore sold only when the farmers are in dire need of money or when there is drought or disease. This has serious implications. When animals are sold to get cash for a pressing need, farmers are willing to accept any price, sometimes well below the market value; they therefore lose substantially. Since they do not receive a fair price for their efforts, they tend to be unwilling to invest in their animals. The other implication is that when animals are sold during drought, or when they are in poor shape as a result of poor nutrition or disease, farmers are unable to demand good prices. There is therefore the need to put in place facilities to help farmers know when to cull animals and how to market their animals. Bring in premium prices To help solve the problems of the market structure, livestock farmers should be encouraged to form livestock producer cooperatives, without political prodding or control. These cooperatives can provide inputs more cheaply (on the basis of economies of scale) and also help in marketing. They could also retain veterinarians on a part-time or full-time basis, which could encourage the privatisation efforts. Such an association could also lobby for fair marketing legislation, especially where monopolies abound. Another recommendation for market restructuring is to introduce payment of premium prices for quality. At the moment there is no price differentiation to encourage production of quality goods that will fetch higher prices. There is an urgent need to restructure the market so that farmers will be paid for quality products and will be more willing to invest in their animals knowing that they will be rewarded with higher prices. Once they receive premium prices, they may be more willing to pay for services like health, knowing that they stand to benefit in the end. The last recommendation is to educate farmers on the need to keep quality animals instead of keeping large numbers for social prestige. This can be done through livestock producer associations and through premium pricing. It may be helpful to work with rural anthropologists/ sociologists to help redirect societal values from quantity to quality. These, it is believed, will help in privatisation of veterinary services in the long term. The opinions expressed in Viewpoint are those of the authors, and do not necessarily reflect the views of CTA.In many developing countries there has been, and still is, a strong move for the government to give up or reduce its involvement in ventures that the market can take care of. The ideological basis for this hinges on the belief that the government..