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.

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.

The most common cause of cystic fibrosis (CF) is deletion of phenylalanine 508 (ΔF508) in the CF transmembrane conductance regulator (CFTR) chloride channel. The ΔF508 mutation produces defects in folding, stability, and channel gating. To identify small-molecule correctors of defective cellular processing, we assayed iodide flux in ΔF508-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 ΔF508-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 ΔF508-CFTR protein. Biochemical studies suggested a mechanism of action involving improved ΔF508-CFTR folding at the ER and stability at the cell surface. The bisaminomethylbithiazoles corrected ΔF508-CFTR in ΔF508/ΔF508 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 ΔF508 mutation.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl⁻ channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with K_i < 10 μM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl⁻ conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant K_i from 1.4 μM at +60 mV to 5.6 μM at −60 mV. Apparent potency was reduced by lowering extracellular Cl⁻ concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (−60 mV holding potential, 5 μM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5–8.0, where it exists predominantly as a monovalent anion with solubility ∼1 mM in water. Topical GlyH-101 (10 μM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 μg) reduced by ∼80% cholera toxin–induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTR_inh-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.