Calcium-activated Chloride Channel TMEM16A Research Articles

Structural and theoretical basis for drug development targeting TMEM16A: Inhibition mechanism of tracheloside analogs

Ion channels play a crucial role in the electrophysiological activities of organisms. The calcium-activated chloride channel TMEM16A is involved in various physiological processes. Therefore, inhibitors of TMEM16A are used to treat diseases caused by TMEM16A dysfunction. However, the unclear inhibition mechanism hinders the progress of drug development. Based on our previous study, we found that the molecular structures of TMEM16A inhibitors tracheloside, matairesinoside and arctigenin are similar. In this study, we conducted a structure-based virtual screening of tracheloside analogs from the PubChem database. The six tracheloside analogs with the highest affinity to TMEM16A were selected, and their inhibitory effects were detected by fluorescence and electrophysiological experiments. Subsequently, the interaction between the tracheloside analogs and TMEM16A was investigated through molecular docking and site-directed mutagenesis. Based on the above results, the mechanism of inhibition of TMEM16A gated conformation by tracheloside analogs was proposed. These findings provide a structural and theoretical basis for drug development targeting TMEM16A.

Niclosamide potentiates TMEM16A and induces vasoconstriction.

The TMEM16A calcium-activated chloride channel is a promising therapeutic target for various diseases. Niclosamide, an anthelmintic medication, has been considered a TMEM16A inhibitor for treating asthma and chronic obstructive pulmonary disease (COPD) but was recently found to possess broad-spectrum off-target effects. Here, we show that, under physiological Ca2+ (200-500 nM) and voltages, niclosamide acutely potentiates TMEM16A. Our computational and functional characterizations pinpoint a putative niclosamide binding site on the extracellular side of TMEM16A. Mutations in this site attenuate the potentiation. Moreover, niclosamide potentiates endogenous TMEM16A in vascular smooth muscle cells, triggers intracellular calcium increase, and constricts the murine mesenteric artery. Our findings advise caution when considering clinical applications of niclosamide as a TMEM16A inhibitor. The identification of the putative niclosamide binding site provides insights into the mechanism of TMEM16A pharmacological modulation and provides insights into developing specific TMEM16A modulators to treat human diseases.

Insights into the function and regulation of the calcium-activated chloride channel TMEM16A

The TMEM16A channel, a member of the TMEM16 protein family comprising chloride (Cl−) channels and lipid scramblases, is activated by the free intracellular Ca2+ increments produced by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release after GqPCRs or Ca2+ entry through cationic channels. It is a ubiquitous transmembrane protein that participates in multiple physiological functions essential to mammals' lives. TMEM16A structure contains two identical 10-segment monomers joined at their transmembrane segment 10. Each monomer harbours one independent hourglass-shaped pore gated by Ca2+ ligation to an orthosteric site adjacent to the pore and controlled by two gates. The orthosteric site is created by assembling negatively charged glutamate side chains near the pore´s cytosolic end. When empty, this site generates an electrostatic barrier that controls channel rectification. In addition, an isoleucine-triad forms a hydrophobic gate at the boundary of the cytosolic vestibule and the inner side of the neck. When the cytosolic Ca2+ rises, one or two Ca2+ ions bind to the orthosteric site in a voltage (V)-dependent manner, thus neutralising the electrostatic barrier and triggering an allosteric gating mechanism propagating via transmembrane segment 6 to the hydrophobic gate. These coordinated events lead to pore opening, allowing the Cl− flux to ensure the physiological response. The Ca2+-dependent function of TMEM16A is highly regulated. Anions with higher permeability than Cl− facilitate V dependence by increasing the Ca2+ sensitivity, intracellular protons can replace Ca2+ and induce channel opening, and phosphatidylinositol 4,5-bisphosphate bound to four cytosolic sites likely maintains Ca2+ sensitivity. Additional regulation is afforded by cytosolic proteins, most likely by phosphorylation and protein-protein interaction mechanisms.

Structural determinants of ligand efficacy in the calcium-activated chloride channel TMEM16A

Structural determinants of ligand efficacy in the calcium-activated chloride channel TMEM16A

Polyunsaturated fatty acids regulate the calcium-activated chloride channel TMEM16A activity

Polyunsaturated fatty acids regulate the calcium-activated chloride channel TMEM16A activity

Mechanistic basis of ligand efficacy in the calcium-activated chloride channel TMEM16A.

Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes the equilibrium between open and closed conformations in a fully ligand-bound state. Calcium-activated chloride channels in the TMEM16 family are important sensors of intracellular calcium signals and are targets for pharmacological modulators, yet a mechanistic understanding of agonist efficacy has remained elusive. Using a combination of cryo-electron microscopy, electrophysiology, and autocorrelation analysis, we now show that agonist efficacy in the ligand-gated channel TMEM16A is dictated by the conformation of the pore-lining helix α6 around the Ca2+ -binding site. The closure of the binding site, which involves the formation of a π-helix below a hinge region in α6, appears to be coupled to the opening of the inner pore gate, thereby governing the channel's open probability and conductance. Our results provide a mechanism for agonist binding and efficacy and a structural basis for the design of potentiators and partial agonists in the TMEM16 family.

Inhibitory effect of daidzein on the calcium-activated chloride channel TMEM16A and its anti-lung adenocarcinoma activity

TMEM16A is highly expressed in a variety of tumor cells and is involved in the growth and metastasis of malignancies. It has been established that down-regulation of TMEM16A expression or functional activity can inhibit tumor cells growth. However, there is a lack of targeted inhibitors with high efficiency and low toxicity. Here, we identified a novel inhibitor daidzein from dozens of natural product molecules. Whole-cell patch clamp data indicated that daidzein inhibits TMEM16A channel in a dose-dependent manner, with IC50 of 1.39 ± 0.59 μM. Western blot result showed that daidzein can also reduce the expression of TMEM16A protein in LA795 cells. These results indicated that the inhibitory effects of daidzein exert on TMEM16A in two ways, both inhibiting TMEM16A current and decreasing its protein expression. In addition, the putative binding sites of daidzein on TMEM16A are G608, G628, and K839 through molecular docking. Moreover, daidzein concentration-dependently reduced cell viability and cell migration, causing G1/S cell cycle arrest in vitro. It was also confirmed that daidzein can effectively inhibit the growth of LA795 lung adenocarcinoma cells implanted nude mice in vivo. In conclusion, daidzein can be used as a lead compound for the development of therapeutic drugs for lung adenocarcinoma.

Identification of a druggable pocket of the calcium-activated chloride channel TMEM16A in its open state

The calcium-activated chloride channel TMEM16A is a potential drug target to treat hypertension, secretory diarrhea, and several cancers. However, all reported TMEM16A structures are either closed or desensitized, and direct inhibition of the open state by drug molecules lacks a reliable structural basis. Therefore, revealing the druggable pocket of TMEM16A exposed in the open state is important for understanding protein-ligand interactions and facilitating rational drug design. Here, we reconstructed the calcium-activated open conformation of TMEM16A using an enhanced sampling algorithm and segmental modeling. Furthermore, we identified an open-state druggable pocket and screened a potent TMEM16A inhibitor, etoposide, which is a derivative of a traditional herbal monomer. Molecular simulations and site-directed mutagenesis showed that etoposide binds to the open state of TMEM16A, thereby blocking the ion conductance pore of the channel. Finally, we demonstrated that etoposide can target TMEM16A to inhibit the proliferation of prostate cancer PC-3cells. Together, these findings provide a deep understanding of the TMEM16A open state at an atomic level and identify pockets for the design of novel inhibitors with broad applications in chloride channel biology, biophysics, and medicinal chemistry.

Oleic acid blocks the calcium-activated chloride channel TMEM16A/ANO1

The calcium-activated chloride channel TMEM16A (ANO1) supports the passive movement of chloride ions across membranes and controls critical cell functions. Here we study the block of wild-type and mutant TMEM16A channels expressed in HEK293 cells by oleic acid, a monounsaturated omega-9 fatty acid beneficial for cardiovascular health. We found that oleic acid irreversibly blocks TMEM16A in a dose- and voltage-dependent manner at low intracellular Ca2+. We tested whether oleic acid interacted with the TMEM16A pore, varying the permeant anion concentration and mutating pore residues. Lowering the permeating anion concentration in the intracellular side did nothing but the blockade was intensified by increasing the anion concentration in the extracellular side. However, the blockade of the pore mutants E633A and I641A was voltage-independent, and the I641A IC50, a mutant with the inner hydrophobic gate in disarray, increased 16-fold. Furthermore, the uncharged methyl-oleate blocked 20–24% of the wild-type and I641A channels regardless of voltage. Our findings suggest that oleic acid inhibits TMEM16A by an allosteric mechanism after the electric field drives oleic acid's charged moiety inside the pore. Block of TMEM16A might be why oleic acid has a beneficial impact on the cardiovascular system.

Paving the way for designing drugs targeting TMEM16A

The calcium-activated chloride channel TMEM16A is involved in several physiological processes and is an important pharmacological target. Dinsdale and colleagues recently unveiled several residues in the outer pore region that constitute a critical site for the design of drugs that modulate TMEM16A channels.

The chloride channel CFTR is not required for cyst growth in an ADPKD mouse model.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of bilateral renal cysts which enlarge continuously, leading to compression of adjacent intact nephrons. The growing cysts lead to a progressive decline in renal function. Cyst growth is driven by enhanced cell proliferation and chloride secretion into the cyst lumen. Chloride secretion is believed to occur mainly by the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR), with some contribution by the calcium-activated chloride channel TMEM16A. However, our previous work suggested TMEM16A as a major factor for renal cyst formation. The contribution of CFTR to cyst formation has never been demonstrated in an adult ADPKD mouse model. We used mice with an inducible tubule-specific Pkd1 knockout, which consistently develop polycystic kidneys upon deletion of Pkd1. Cellular properties, ion currents, and cyst development in these mice were compared with that of mice carrying a co-deletion of Pkd1 and Cftr. Knockout of Cftr did not reveal any significant impact on cyst formation in the ADPKD mouse model. Furthermore, knockout of Cftr did not attenuate the largely augmented cell proliferation observed in Pkd1 knockout kidneys. Patch clamp analysis on primary renal epithelial cells lacking expression of Pkd1 indicated an only marginal contribution of CFTR to whole cell Cl- currents, which were clearly dominated by calcium-activated TMEM16A currents. In conclusion, CFTR does not essentially contribute to renal cyst formation in mice caused by deletion of Pkd1. Enhanced cell proliferation and chloride secretion is caused primarily by upregulation of the calcium-activated chloride channel TMEM16A.

The Calcium-Activated Chloride Channel TMEM16A is Inhibitied by Liquiritigenin.

The transmembrane 16 (TMEM16) family contains 10 subtypes, and the function of each protein is different. TMEM16A is a calcium-activated chloride channel involved in physiological and pathological situations. Liquiritigenin is an aglycone derived from Glycyrrhiza glabra, and it is generated via the metabolism of enterobacterial flora. It has been known that liquiritigenin reduces pain sensation involving TMEM16A activation in primary sensory neurons. In addition, other pharmacological effects of liquiritigenin in physiological functions involving TMEM16A have been reported. However, the relationship between TMEM16A and liquiritigenin is still unknown. Therefore, we hypothesized that TMEM16A is inhibited by liquiritigenin. To confirm this hypothesis, we investigated the effect of liquiritigenin on TMEM16A currents evoked by intracellular free calcium in HEK293T cells transfected with TMEM16A. In this study, we found that liquiritigenin inhibited the mouse and human TMEM16A currents. To further confirm its selectivity, we also investigated its pharmacological effects on other ion channels, including transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), which are non-selective cation channels involved in pain sensation. However, liquiritigenin did not inhibit the currents of TRPV1 and TRPA1 induced by capsaicin and allyl isothiocyanate, respectively. Therefore, our findings indicate that selective TMEM16A inhibition could be one molecular mechanism that explains liquiritigenin-induced pain reduction. Additionally, we also investigated the inhibitory effects of estrogens on TMEM16A because liquiritigenin reportedly binds to the estrogen receptor. In this study, a pregnancy-dependent estrogen, estriol, significantly inhibited TMEM16A. However, the efficacy was weak. Although there is a possibility that TMEM16A activity could be suppressed during pregnancy, the physiological significance seems to be small. Thus, the inhibitory effect of estrogen might not be significant under physiological conditions. Furthermore, we investigated the effect of dihydrodaidzein, which is an analog of liquiritigenin that has a hydroxyphenyl at different carbon atom of pyranose. Dihydrodaidzein also inhibited mouse and human TMEM16A. However, the inhibitory effects were weaker than those of liquiritigenin. This suggests that the efficacy of TMEM16A antagonists depends on the hydroxyl group positions. Our finding of liquiritigenin-dependent TMEM16A inhibition could connect the current fragmented knowledge of the physiological and pathological mechanisms involving TMEM16A and liquiritigenin.

Mechanism of pore opening in the calcium-activated chloride channel TMEM16A

The anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation.

Gating the pore of the calcium-activated chloride channel TMEM16A

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.

Structures of a Constitutively Active Mutant of the Calcium-Activated Chloride Channel TMEM16A in Ligand-Free and -Bound States

The binding of Ca2+ from the cytoplasm to the anion-selective channel TMEM16A triggers a conformational change around its binding site, which is coupled to the opening of an hourglass-shaped pore. We have previously identified three bulky hydrophobic residues at the intracellular entrance of the neck as constituents of the gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. Here, we determined cryo-electron microscopy structures of one of these constitutively active mutants in the absence and presence of Ca2+. Whereas the mutant resembles wild-type in the Ca2+-bound state, the Ca2+-free structure exhibits pronounced differences in the pore region compared to the corresponding structure of the wild-type channel. Similar to wild-type, the lower half of the Ca2+-binding and pore-lining helix α6 in the mutant adopts a ‘down’ conformation and directly coordinates the bound Ca2+ in the Ca2+-bound state. In the absence of Ca2+, this helix likewise dissociates from the binding site in the mutant, but the movement is less pronounced and proceeds in a different direction compared to wild-type. Consequently, α6 in the mutant remains in a position that is closer to its fully activated conformation, thereby lowering the energetic penalty for channel opening. Despite the difference in its position, the relaxation of α6 from a π- to an α-helix upon dissociation of Ca2+ is also observed in the mutant structures, suggesting that pore opening can proceed without a transition into the more strained π-helix conformation. Together, these observations emphasize a coupling between the gate and the Ca2+ binding element of α6, allowing Ca2+ binding to stabilize a conductive state of the channel, and provide a structural view of ligand-free activation of TMEM16A.

Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction.

The calcium‐activated chloride channel (CaCC) TMEM16A enables chloride secretion across several transporting epithelia, including in the airways. Additional roles for TMEM16A have been proposed, which include regulating mucus production and secretion and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, could affect any of these proposed biological roles in the airways. In vitro, neither a potent and selective TMEM16A potentiator (ETX001) nor the potent TMEM16A inhibitor (Ani9) influenced either baseline mucin release or goblet cell numbers in well‐differentiated primary human bronchial epithelial (HBE) cells. In vivo, a TMEM16A potentiator was without effect on goblet cell emptying in an IL‐13 stimulated goblet cell metaplasia model. Using freshly isolated human bronchi and pulmonary arteries, neither ETX001 or Ani9 had any effect on the contractile or relaxant responses of the tissues. In vivo, ETX001 also failed to influence either lung or cardiovascular function when delivered directly into the airways of telemetered rats. Together, these studies do not support a role for TMEM16A in the regulation of goblet cell numbers or baseline mucin release, or on the regulation of airway or pulmonary artery smooth muscle contraction.

Cobalt ion interaction with TMEM16A calcium-activated chloride channel: Inhibition and potentiation.

TMEM16A, a Ca2+-sensitive Cl- channel, plays key roles in many physiological functions related to Cl- transport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-μM to low μM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of μM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.

Stepwise Dissociation of an Inner Gate Controls Pore Opening in the Calcium-activated Chloride Channel TMEM16A

TMEM16A is a Ca2+-activated Cl- channel that plays key roles in diverse physiological processes. This channel functions as a homodimer with each subunit containing an independent pore and closely apposed Ca2+ binding sites. Recent cryo-electron microscopy structures of TMEM16A have revealed a conformational change involving the rearrangement of a pore-lining helix (α6) upon Ca2+ binding in a process that precedes pore activation. We have further shown that an electrostatic gating mechanism operates in TMEM16A where bound Ca2+ ions lower the energy barriers for anion conduction via direct Coulombic interactions. However, despite the described advances, the location of the gate that prevents ion conduction in the close conformation has remained elusive. Here, we investigated conformational gating of TMEM16A using electrophysiology and spectral analysis. Systematic mutagenesis of pore-lining helices identifies a specific cluster of hydrophobic residues at the inner pore that closes the anion conduction path in the closed state. The stability of the cluster increases with hydrophobic volume but correlates inversely with increasing hydrophilicity, consistent with a gate region where water and ions are excluded when closed. We analysed the power spectra of alanine mutants at the constriction and found that the loss of hydrophobic volume results in enhanced pore opening at equilibrium. The presence of three Lorentzian components in the spectra suggests an intermediate gating step that relays α6 activation to the opening of the pore. Combined with analysis of multiple double-mutant cycles, our results reveal a pore opening mechanism that involves stepwise dissociation of the inner hydrophobic gate and the pairwise interactions underlying these conformational transitions.

A Network of PIP2 Binding Sites Regulate Gating of the Calcium-Activated Chloride Channel TMEM16A

TMEM16A is a Ca2+-activated Cl− channel that regulates diverse cellular functions including fluid secretion, neuronal excitability and smooth muscle contraction. TMEM16A is activated by cytosolic Ca2+ and modulated by binding of the signaling lipid, PIP2. However, it remains elusive how PIP2 binds the channel and how the binding affects molecular events underlying transport in the protein. Here, we use extended molecular dynamics (MD) simulations coupled with electrophysiology, mutagenesis, and functional assays, to characterize PIP2 binding modes and sites in TMEM16A and the conformational response of the channel to PIP2 binding.

An Allosteric Gating Mechanism of TMEM16A Calcium-Activated Chloride Channel

The Ca2+-activated chloride channel (CaCC) TMEM16A is widely expressed throughout the body with diverse functions ranging from trans-epithelial fluid transport, smooth muscle contraction, to nociception. Activation of TMEM16A requires binding of intracellular Ca2+ whereas binding of the phosphatidylinositol PIP2 facilitates channel opening. Assembled as a homodimer, each TMEM16A monomer is consisted of ten transmembrane (TM) segments in which TMs 3 to 8 form an independent chloride-permeable pore. Owing to their direct contributions to the permeation pathway, TMs 3 to 8 have been extensively studied. Our recent work suggests that TMs 3-8 comprise of two functionally distinct modules in which TMs 6-8 constitute the Ca2+-binding module that controls Ca2+-dependent channel activation while TMs 3-5 form a regulatory module that mediates PIP2-dependent modulation of channel opening. However, it remains unknown whether the remaining TMs, namely TMs 1, 2, 9, and 10, all of which are distant from the permeation pathway, also contribute to channel gating. Here we combine electrophysiological analyses with mutagenesis to demonstrate that TM2 and TM10 interaction allosterically controls TMEM16A gating. These studies not only provide the molecular underpinnings of allosteric regulatory mechanism in TMEM16A but will also facilitate our mechanistic understanding of the gating mechanism in TMEM16 Ca2+-activated ion channels and lipid scramblases.