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Various human tissues express the calcium-activated chloride channel Anoctamin 1 (ANO1), also known as TMEM16A. ANO1 allows the passive chloride flux that controls different physiological functions ranging from muscle contraction, fluid and hormone secretion, gastrointestinal motility, and electrical excitability. Overexpression of ANO1 is associated with pathological conditions such as hypertension and cancer. The molecular cloning of ANO1 has led to a surge in structural, functional, and physiological studies of the channel in several tissues. ANO1 is a homodimer channel harboring two pores - one in each monomer - that work independently. Each pore is activated by voltage-dependent binding of two intracellular calcium ions to a high-affinity-binding site. In addition, the binding of phosphatidylinositol 4,5-bisphosphate to sites scattered throughout the cytosolic side of the protein aids the calcium activation process. Furthermore, many pharmacological studies have established ANO1 as a target of promising compounds that could treat several illnesses. This chapter describes our current understanding of the physiological roles of ANO1 and its regulation under physiological conditions as well as new pharmacological compounds with potential therapeutic applications.

Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl− channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.

Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure–function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate–gating relationship in TMEM16A remain less explored. Here, we study the gating–permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN− mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN−. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

"TMEM16A es un canal de cloruro activado por calcio que controla el transporte de líquido epitelial, la contracción del músculo liso, el bloqueo de la poliespermia y la liberación de insulina, entre otros. La activación de TMEM16A es un mecanismo complejo que requiere incrementos de calcio intracelular y despolarizaciones de la membrana plasmática. TMEM16A no tiene un sensor de voltaje canónico, sin embargo, puede ser activado por la diferencia de voltaje a través de la membrana, aunque el mecanismo aún no está claro. En este trabajo mostramos que el canal TMEM16A silvestre se activa por voltaje en ausencia de calcio. Las corrientes muestran una fuerte rectificación saliente, la activación es muy rápida (

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.