Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

The most common cause of cystic fibrosis (CF) is deletion of phenylalanine 508 (∆F508) in the CF transmembrane conductance regulator (CFTR) chloride channel. The ∆F508 mutation produces defects in folding, stability, and channel gating. To identify small-molecule correctors of defective cellular processing, we assayed iodide flux in ∆F508-CFTR-transfected epithelial cells using a fluorescent halide indicator. Screening of 150,000 chemically diverse compounds and more than 1,500 analogs of active compounds yielded several classes of ∆F508-CFTR correctors (aminoarylthiazoles, quinazolinylaminopyrimidinones, and bisaminomethylbithiazoles) with micromolar potency that produced greater apical membrane chloride current than did low-temperature rescue. Correction was seen within 3-6 hours and persisted for more than 12 hours after washout. Functional correction was correlated with plasma membrane expression of complex-glycosylated ∆F508-CFTR protein. Biochemical studies suggested a mechanism of action involving improved ∆F508-CFTR folding at the ER and stability at the cell surface. The bisaminomethylbithiazoles corrected ∆F508-CFTR in ∆F508/∆F508 human bronchial epithelia but did not correct a different temperature-sensitive CFTR mutant (P574H-CFTR) or a dopamine receptor mutant. Small-molecule correctors may be useful in the treatment of CF caused by the ∆F508 mutation.

TMEM16 proteins, also known as anoctamins, are involved in a variety of functions that include ion transport, phospholipid scrambling, and regulation of other membrane proteins. The first two members of the family, TMEM16A (anoctamin-1, ANO1) and TMEM16B (anoctamin-2, ANO2), function as Ca2+-activated Cl- channels (CaCCs), a type of ion channel that plays important functions such as transepithelial ion transport, smooth muscle contraction, olfaction, phototransduction, nociception, and control of neuronal excitability. Genetic ablation of TMEM16A in mice causes impairment of epithelial Cl- secretion, tracheal abnormalities, and block of gastrointestinal peristalsis. TMEM16A is directly regulated by cytosolic Ca2+ as well as indirectly by its interaction with calmodulin. Other members of the anoctamin family, such as TMEM16C, TMEM16D, TMEM16F, TMEM16G, and TMEM16J, may work as phospholipid scramblases and/or ion channels. In particular, TMEM16F (ANO6) is a major contributor to the process of phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane. Intriguingly, TMEM16F is also associated with the appearance of anion/cation channels activated by very high Ca2+ concentrations. Furthermore, a TMEM16 protein expressed in Aspergillus fumigatus displays both ion channel and lipid scramblase activity. This finding suggests that dual function is an ancestral characteristic of TMEM16 proteins and that some members, such as TMEM16A and TMEM16B, have evolved to a pure channel function. Mutations in anoctamin genes (ANO3, ANO5, ANO6, and ANO10) cause various genetic diseases. These diseases suggest the involvement of anoctamins in a variety of cell functions whose link with ion transport and/or lipid scrambling needs to be clarified.

Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABA(A) (gamma-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery.

The green fluorescent protein YFP-H148Q is sensitive to halides by a mechanism involving halide binding and a shift in pK a . However, a limitation of YFP-H148Q is its low halide sensitivity, with K d s 100 mM for Cl 3 . Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement of Cl 3 concentration in subcellular organelles. YFP-H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated. An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clones revealed improved anion sensitivities with K d down to 2 mM for I 3 (I152L), 40 mM for Cl 3 (V163S), and 10 mM for NO 3 3 (I152L). The anion-sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel. ß

-activated Cl Ϫ channels (CaCCs) 2 play a major physiological role in various types of cells and tissues (1-4). In epithelial cells, opening of CaCCs in the apical membrane generates a flux of Cl Ϫ that drives transepithelial water transport. The resulting CaCC-dependent electrolyte/fluid secretion is one of the mechanisms responsible for exocrine secretion in many types of glands and for hydration of the airway surface (2). In particular, the activity of CaCC and of the cystic fibrosis transmembrane conductance regulator chloride channel controls the thickness of the periciliary fluid that is important for optimal mucociliary clearance (5, 6). Deficit in cystic fibrosis transmembrane conductance regulator activity, which occurs in cystic fibrosis, favors bacterial colonization of the airways. Under such conditions, activity of CaCCs may be important in compensating, at least partially, the defect in Cl Ϫ transport. In smooth muscle cells, activation of CaCCs is important in the process of contraction (1,7,8). CaCCs are also present in olfactory receptors, in dorsal root ganglion neurones, and in oocytes (3, 4, 9, 10). The biophysical properties of CaCCs are not homogeneous among different cells and tissues. In many cases, CaCCs are activated by Ca 2ϩ in a wide range of concentrations between 0.06 and 1 M (11-14) and are also voltage-dependent, with membrane depolarization increasing the activity (7, 10 -16). However, CaCCs that need much higher Ca 2ϩ concentration (9, 17, 18) and that are devoid of voltage dependence are also known (9,17,19). Moreover, the mechanism of regulation by Ca 2ϩ is unclear. In some studies, Ca 2ϩ seems to activate the channels through calmodulin (20). In others, activation requires the intervention of a Ca 2ϩ /calmodulin-dependent kinase (19,21). Finally, there are cell types where phosphorylation has actually an inhibitory effect on CaCCs (7,8,14). The differences may be due to heterogeneity of the proteins that actually constitute the CaCCs.Until recently, the molecular identity of CaCCs was a controversial issue, with ClCA proteins, ClC3, and bestrophins being postulated as possible candidates (1,22). Three recent studies, including one from our laboratory, have identified the TMEM16A protein (also known as ANO-1) as a probable . TMEM16A is a membrane protein with eight putative transmembrane segments belonging to a family including other nine members (TMEM16B-K). In our previous study (23), we identified several TMEM16A transcripts probably generated by selection of alternative splice sites. The alternative sequences coded for protein segments that we named a (116 residues), b (22 residues), c (4 residues), and d (26 residues). The former two segments are localized in the N terminus, whereas the latter two segments are localized in the first intracellular loop.A TMEM16A mouse knock-out model has been also generated. The phenotype of these animals is severe and characterized by altered formation of tracheal cartilage rings (26). This alteration, causing airway collapse, may be resp...

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

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