Channel TMEM16A Research Articles

Targeting TMEM16A to reverse vasoconstriction and remodelling inidiopathic pulmonary arterial hypertension.

Our systematic analysis of anion channels and transporters in idiopathic pulmonary arterial hypertension (IPAH) showed marked upregulation of the Cl- channel TMEM16A gene. We hypothesised that TMEM16A overexpression might represent a novel vicious circle in the molecular pathways causing pulmonary arterial hypertension (PAH).We investigated healthy donor lungs (n=40) and recipient lungs with IPAH (n=38) for the expression of anion channel and transporter genes in small pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs).In IPAH, TMEM16A was strongly upregulated and patch-clamp recordings confirmed an increased Cl- current in PASMCs (n=9-10). These cells were depolarised and could be repolarised by TMEM16A inhibitors or knock-down experiments (n=6-10). Inhibition/knock-down of TMEM16A reduced the proliferation of IPAH-PASMCs (n=6). Conversely, overexpression of TMEM16A in healthy donor PASMCs produced an IPAH-like phenotype. Chronic application of benzbromarone in two independent animal models significantly decreased right ventricular pressure and reversed remodelling of established pulmonary hypertension.Our findings suggest that increased TMEM16A expression and activity comprise an important pathologic mechanism underlying the vasoconstriction and remodelling of pulmonary arteries in PAH. Inhibition of TMEM16A represents a novel therapeutic approach to reverse remodelling in PAH.

Inhibition of TMEM16A by Docosahexaenoic Acid Plays a Crucial Role in Blood Vessel Relaxation

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid with beneficial cardiovascular effects, such as vasodilation. The mechanism underlying this vasodilation, however, is not fully defined. In arterial smooth muscle, TMEM16A chloride channels promote membrane depolarisation and vessel contraction. Cloned TMEM16A channels are inhibited by DHA. Here, we examine whether inhibition of native TMEM16A by DHA plays a role in aortic relaxation. Patch-clamp electrophysiology and isometric tension recordings were utilised to study the effect of DHA on cloned TMEM16A channels and aortic contractility. DHA inhibited TMEM16A currents in a dose dependent manner, IC50=15.4±1.5 μM (n=10). To study the regulation of TMEM16A by DHA in response to agonist-induced increase in [Ca2+]i, TMEM16A was co-expressed with the α1A-adrenoreceptor and currents recorded in response to 1 μM phenylephrine (PE). In the absence and presence of DHA (10 μM) the TMEM16A current increased by 7.6±1.8 fold (n=12) and 2.8±0.5 fold (n=6), respectively (p<0.05). In aortic rings, inhibition of TMEM16A with either 70 μM DHA or 1 μM 2-(4-Chloro-2-methylphenoxy)-N′-(2-methoxybenzylidene)acetohydrazide (Ani9) reduced PE-induced aortic contraction by 1.9±0.4 fold (n=9) and 2.0±0.4 fold (n=7), respectively. Inhibition of the NKCC1 transporter with 40 μM bumetanide, to lower intracellular Cl- concentration ([Cl-]), reduced aortic response to 1 μM PE by 3.3±0.4 fold (n=10). Conversely, lowering extracellular [Cl-] increased the aortic response to 1 μM PE by 9.3±3.4 fold (n=5); this effect was reduced to 2.8±1.0 fold (n=5) in the presence of 70 μM DHA (p<0.05), implicating TMEM16A channels in the response. DHA-mediated inhibition of TMEM16A channels reduces aortic contractility. This study highlights TMEM16A as a target for modulating vascular tone. Identification of the TMEM16A DHA binding site may enable development of new small molecules aiming to reduce blood pressure.

Cryo-EM Structures Reveal Bilayer Remodeling during Ca2+ Activation of a TMEM16 Scramblase

The plasma membranes of eukaryotic cells are organized asymmetrically for cellular function. At rest, polar and charged lipids are sequestered to the inner leaflet by the activity of ATP-driven pumps. Activation of a specialized class of membrane proteins – phospholipid scramblases – causes rapid collapse of this asymmetry and externalization of negatively charged phosphatidylserine molecules. As a result, extracellular signaling networks, controlling processes such as apoptosis, blood coagulation, membrane fusion and repair, are activated. The TMEM16 family of membrane proteins includes phospholipid scramblases and Cl- channels, all of which are Ca2+-dependent. Prior structural and functional analyses of the fungal TMEM16 scramblase from Nectria haematococca identified a membrane-exposed hydrophilic groove that serves as the lipid translocation pathway. In the TMEM16A channel, this pathway is sealed from the membrane, thus preventing lipid access and enabling ion permeation. However, the mechanisms underlying Ca2+-dependent gating of TMEM16 scramblases, and their effects on the surrounding lipid bilayer, remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. Differences between the Ca2+-free and Ca2+-bound states reveal that Ca2+ binding induces a global rearrangement of the transmembrane and cytosolic regions, which causes the lipid pathway to open. Further, comparison of these structures and a third in the presence of a long-chain inhibitory lipid, together with functional experiments, reveal that scramblases cause profound remodeling of the surrounding membrane. Specifically, scramblase activation causes the bilayer to thin at the open lipid pathway and the outer leaflet to bend towards the center of the bilayer at the dimer interface. We propose a model in which trans-bilayer lipid movement is enabled by these membrane and protein rearrangements and provide a general mechanistic framework for phospholipid scrambling.

TMEM16A in Cystic Fibrosis: Activating or Inhibiting?

The inflammatory airway disease cystic fibrosis (CF) is characterized by airway obstruction due to mucus hypersecretion, airway plugging, and bronchoconstriction. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is dysfunctional in CF, leading to defects in epithelial transport. Although CF pathogenesis is still disputed, activation of alternative Cl− channels is assumed to improve lung function in CF. Two suitable non-CFTR Cl− channels are present in the airway epithelium, the Ca2+ activated channel TMEM16A and SLC26A9. Activation of these channels is thought to be feasible to improve hydration of the airway mucus and to increase mucociliary clearance. Interestingly, both channels are upregulated during inflammatory lung disease. They are assumed to support fluid secretion, necessary to hydrate excess mucus and to maintain mucus clearance. During inflammation, however, TMEM16A is upregulated particularly in mucus producing cells, with only little expression in ciliated cells. Recently it was shown that knockout of TMEM16A in ciliated cells strongly compromises Cl− conductance and attenuated mucus secretion, but does not lead to a CF-like lung disease and airway plugging. Along this line, activation of TMEM16A by denufosol, a stable purinergic ligand, failed to demonstrate any benefit to CF patients in earlier studies. It rather induced adverse effects such as cough. A number of studies suggest that TMEM16A is essential for mucus secretion and possibly also for mucus production. Evidence is now provided for a crucial role of TMEM16A in fusion of mucus-filled granules with the apical plasma membrane and cellular exocytosis. This is probably due to local Ca2+ signals facilitated by TMEM16A. Taken together, TMEM16A supports fluid secretion by ciliated airway epithelial cells, but also maintains excessive mucus secretion during inflammatory airway disease. Because TMEM16A also supports airway smooth muscle contraction, inhibition rather than activation of TMEM16A might be the appropriate treatment for CF lung disease, asthma and COPD. As a number of FDA-approved and well-tolerated drugs have been shown to inhibit TMEM16A, evaluation in clinical trials appears timely.

Lipid Peroxidation Drives Renal Cyst Growth In Vitro through Activation of TMEM16A.

Transepithelial chloride- secretion, through the chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1), drives cyst enlargement in polycystic kidney disease (PKD). Polycystic kidneys are hypoxic, and oxidative stress activates TMEM16A. However, mechanisms for channel activation in PKD remain obscure. Using tissue samples from patients with autosomal dominant PKD, embryonic kidney cultures, and an MDCK in vitro cyst model, we assessed peroxidation of plasma membrane phospholipids in human and mouse polycystic kidneys. We also used electrophysiologic Ussing chamber and patch clamp experiments to analyze activation of TMEM16A and growth of renal cysts. Peroxidation of phospholipids in human and mouse kidneys as well as MDCK cysts in vitro is probably due to enhanced levels of reactive oxygen species. Lipid peroxidation correlated with increased cyst volume as shown in renal cultures and MDCK cysts in three-dimensional cultures. Reactive oxygen species and lipid peroxidation strongly activated TMEM16A, leading to depletion of calcium ion stores and store-operated calcium influx. Activation of TMEM16A- and CFTR-dependent chloride secretion strongly augmented cyst growth. Exposure to scavengers of reactive oxygen species, such as glutathione, coenzyme Q10, or idebenone (a synthetic coenzyme Q10 homolog), as well as inhibition of oxidative lipid damage by ferrostatin-1 largely reduced activation of TMEM16A. Inhibition of TMEM16A reduced proliferation and fluid secretion in vitro. These findings indicate that activation of TMEM16A by lipid peroxidation drives growth of renal cysts. We propose direct inhibition of TMEM16A or inhibition of lipid peroxidation as potentially powerful therapeutic approaches to delay cyst development in PKD.

The Natural Compound Cinnamaldehyde is a Novel Activator of Calcium-Activated Chloride Channel.

Calcium-activated chloride channels (CaCCs) play important roles in a multitude of physiological processes, and in many cells types, TMEM16A was identified as the molecular basis of CaCC. Abnormal CaCC function has been implicated in variety of diseases, which reinforces the need for modulators of CaCCs/TMEM16A. However, there are few specific, clinical modulators of CaCCs. Here, we identified a potent novel activator of TMEM16A from a bank of traditional Chinese medicines (TCM) and explored its mechanism of activation by laser confocal scanning microscopy and patch clamping. Fluorescence data demonstrated that among the 36 tested TCM medicines, one compound, cinnamaldehyde (CA), can activate the TMEM16A channel in a dose-dependent manner. To determine the mechanism by which CA activates the TMEM16A channel, we performed an excised patch clamp experiment and measured the intracellular calcium concentration in fluorescence experiments. Our data show that CA activates TMEM16A channels by elevating the intracellular concentrations of calcium ions. The results of the whole-cell patch clamping showed that CA dose-dependently activates these channels, with an EC50 of 9.73 ± 5.64µM at + 80mV, and prolongs the deactivation of TMEM16A. Finally, we found that CA can strengthen contractions of the ileum in guinea pigs by activating TMEM16A. The results demonstrate that CA is a novel, natural activator of TMEM16A.

Matrine is a novel inhibitor of the TMEM16A chloride channel with antilung adenocarcinoma effects.

Calcium-activated chloride channels (CaCCs) are ion channels with key roles in physiological processes. They are abnormally expressed in various cancers, including esophageal squamous cell cancer, head and neck squamous cell carcinoma, colorectal cancer, and gastrointestinal stromal tumors. The CaCC component TMEM16A/ANO1 was recently shown to be overexpressed in lung adenocarcinoma cells and may serve as a tumorigenic protein. In this study, we determined that matrine is a potent TMEM16A inhibitor that exerts anti-lung adenocarcinoma effects. Patch clamp experiments showed that matrine inhibited TMEM16A current in a concentration-dependent manner with an IC 50 of 27.94 ± 4.78 μM. Molecular simulation and site-directed mutation experiments demonstrated that the matrine-sensitive sites of the TMEM16A channel involve the amino acids Y355, F411, and F415. Results of cell viability and wound healing assays showed that matrine significantly inhibited the proliferation and migration of LA795 cells, which exhibit high TMEM16A expression. In contrast, matrine has only weak inhibitory effect on CCD-19Lu and HeLa cells lacking TMEM16A expression. Matrine-induced effects on the proliferation and migration of LA795 cells were abrogated upon shRNA-mediated TMEM16A knockdown in LA795 cells. Finally, in vivo experiments demonstrated that matrine dramatically inhibited the growth of lung adenocarcinoma xenograft tumors in mice but did not affect mouse body weight. Collectively, these data indicate that matrine is an effective and safe TMEM16A inhibitor and that TMEM16A is the target of matrine anti-lung adenocarcinoma activity. These findings provide new insight for the development of novel treatments for lung adenocarcinoma.

Calcium-dependent electrostatic control of anion access to the pore of the calcium-activated chloride channel TMEM16A.

TMEM16A is a ligand-gated anion channel that is activated by intracellular Ca2+. This channel comprises two independent pores and closely apposed Ca2+ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (Paulino et al., 2017a). Here, we demonstrate the electrostatic control of permeation by the bound calcium ions in mouse TMEM16A using electrophysiology and Poisson-Boltzmann calculations. The currents of constitutively active mutants lose their outward rectification as a function of Ca2+ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca2+ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca2+ binding to enable an otherwise sub-conductive pore to conduct with full capacity.

Intraluminal pressure triggers myogenic response via activation of calcium spark and calcium-activated chloride channel in rat renal afferent arteriole.

Myogenic contraction of renal arterioles is an important regulatory mechanism for renal blood flow autoregulation. We have previously demonstrated that integrin-mediated mechanical force increases the occurrence of Ca2+ sparks in freshly isolated renal vascular smooth muscle cells (VSMCs). To further test whether the generation of Ca2+ sparks is a downstream signal of mechanotransduction in pressure-induced myogenic constriction, the relationship between Ca2+ sparks and transmural perfusion pressure was investigated in intact VSMCs of pressurized rat afferent arterioles. Spontaneous Ca2+ sparks were found in VSMCs when afferent arterioles were perfused at 80 mmHg. The spark frequency was significantly increased when perfusion pressure was increased to 120 mmHg. A similar increase of spark frequency was also observed in arterioles stimulated with β1-integrin-activating antibody. Moreover, spark frequency was significantly higher in arterioles of spontaneous hypertensive rats at 80 and 120 mmHg. Spontaneous membrane current recorded using whole cell perforated patch in renal VSMCs showed predominant activity of spontaneous transient inward currents instead of spontaneous transient outward currents when holding potential was set close to physiological resting membrane potential. Real-time PCR and immunohistochemistry confirmed the expression of Ca2+-activated Cl- channel (ClCa) TMEM16A in renal VSMCs. Inhibition of TMEM16A with T16Ainh-A01 impaired the pressure-induced myogenic contraction in perfused afferent arterioles. Our study, for the first time to our knowledge, detected Ca2+ sparks in VSMCs of intact afferent arterioles, and their frequencies were positively modulated by the perfusion pressure. Our results suggest that Ca2+ sparks may couple to ClCa channels and trigger pressure-induced myogenic constriction via membrane depolarization.

Diltiazem inhibits proliferation and motility of hepatocellular cells in vitro by downregulating calcium-activated chloride channel TMEM16A

To assess the inhibitory effect of diltiazem, a calcium channel inhibitor, on the proliferation and mobility of human hepatocellular carcinoma cells in vitro and explore the possible mechanism. Two human hepatocellular carcinoma cell lines, MHCC97H and 7402, were treated with different concentrations (0-400 μmol/L) of diltiazem for 12, 24, or 48 h, and the changes in the cell proliferation and mobility were observed with MTT assay and wound healing assay, respectively. The changes in the expressions of calcium-activated chloride channel TMEM16A at mRNA and protein levels in the treated cells were detected using RT-PCR and immunocytochemistry. Treatment with diltiazem obviously inhibited the proliferation and suppressed the mobility of MHCC97H and 7402 cells in a time- and concentration-dependent manner (P < 0.05). Treatment with 100 μmol/L diltiazem for 24 h significantly inhibited the proliferation of MHCC97H cells and down-regulated the mRNA and protein levels of TMEM16A. In 7402 cells, diltiazem treatment at 50 μmol/L for 48 h resulted in the most significant inhibitory effect on the cell proliferation and TMEM16A expressions. Diltiazem can transiently inhibit the invasion of hepatocellular carcinoma cells in vitro possibly by down-regulating the expression of TMEM16A at both the mRNA and protein levels.

The TMEM16A channel mediates the fast polyspermy block in Xenopus laevis.

In externally fertilizing animals, such as sea urchins and frogs, prolonged depolarization of the egg immediately after fertilization inhibits the entry of additional sperm-a phenomenon known as the fast block to polyspermy. In the African clawed frog Xenopus laevis, this depolarization is driven by Ca2+-activated Cl- efflux. Although the prominent Ca2+-activated Cl- currents generated in immature X. laevis oocytes are mediated by X. laevis transmembrane protein 16a (xTMEM16A) channels, little is known about the channels that contribute to the fast block in mature eggs. Moreover, the gamete undergoes a gross transformation as it develops from an immature oocyte into a fertilization-competent egg. Here, we report the results of our approach to identify the Ca2+-activated Cl- channel that triggers the fast block. By querying published proteomic and RNA-sequencing data, we identify two Ca2+-activated Cl- channels expressed in fertilization-competent X. laevis eggs: xTMEM16A and X. laevis bestrophin 2A (xBEST2A). By exogenously expressing xTMEM16A and xBEST2A in axolotl cells lacking endogenous Ca2+-activated currents, we characterize the effect of inhibitors on currents mediated by these channels. None of the inhibitors tested block xBEST2A currents specifically. However, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) and N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) each reduce xTMEM16A currents by more than 70% while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we find that Ani9 and MONNA effectively diminish fertilization-evoked depolarizations. Additionally, these inhibitors lead to increased polyspermy in X. laevis embryos. These results indicate that fertilization activates TMEM16A channels in X. laevis eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.

CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A.

The Chloride Channel Accessory (CLCA) protein family was first characterized as regulators of calcium-activated chloride channel (CaCC) currents (ICaCC), but the mechanism has not been fully established. We hypothesized that CLCAs might regulate ICaCC by modulating intracellular calcium levels. In cells stably expressing human CLCA2 or vector, we found by calcium imaging that CLCA2 moderately enhanced intracellular-store release but dramatically increased store-operated entry of calcium upon cytosolic depletion. Moreover, another family member, CLCA1, produced similar effects on intracellular calcium mobilization. Co-immunoprecipitation revealed that CLCA2 interacted with the plasma membrane store-operated calcium channel ORAI-1 and the ER calcium sensor STIM-1. The effect of CLCA2 on ICaCC was tested in HEK293 stably expressing calcium-activated chloride channel TMEM16A. Co-expression of CLCA2 nearly doubled ICaCC in response to a calcium ionophore. These results unveil a new mechanism by which CLCA family members activate ICaCC and suggest a broader role in calcium-dependent processes.

Regulation of Intracellular Calcium in Uterine Leiomyomas

BackgroundUterine leiomyomas (fibroids) are benign smooth muscle cell tumors of the uterus that affect a large percentage of reproductive aged women, contribute to infertility, excessive menstrual bleeding, and represent the leading cause of hysterectomy (~70%). Despite their importance, the mechanisms that promote fibroid growth are still not well‐understood. Fibroids are comprised of smooth muscle cells (SMCs) and fibroblasts and their growth is modulated by hormones including estrogen and progesterone. Intracellular calcium [Ca2+]i signaling is a key aspect of SMC physiology in promoting contractility, cell proliferation, and fibrosis: aspects relevant to fibroid biology. While [Ca2+]i regulation in normal myometrium is well characterized, this has not been explored in fibroids. The goal of this study was to explore [Ca2+]i regulation in fibroid SMCs and the effect of sex steroids towards obtaining insights into tumor progression.MethodsCells were enzymatically isolated from normal myometrium and select fibroids of different sizes from patients undergoing myomectomy at Mayo Clinic, Rochester, MN (IRB approved). [Ca2+]i was measured using fura‐2/AM and real‐time fluorescence microscopy. The contribution of Ca2+ influx vs. intracellular pathways in the responses to 1uM oxytocin were assessed using pharmacological inhibitors/activators. Cells were pretreated with vehicle, 1nM estrogen, or 1nM progesterone to assess the effect of sex steroids.ResultsFibroid‐derived cells showed a greater contribution of Ca2+ influx pathways in their responses to oxytocin than normal myometrium. Here, the Ca2+‐activated chloride channel TMEM16A (Ano1) was found to be contributory, with Western analysis showing higher levels of Ano1 in fibroids. Exposure to estrogen resulted in significantly higher [Ca2+]i responses compared to cells treated with vehicle or progesterone, although progesterone did enhance [Ca2+]i responses as well. Estrogen increased Ano1 expression. Cell proliferation was also increased in fibroid cells.ConclusionsExposure to estrogen and progesterone enhances oxytocin induced [Ca2+]i responses of fibroid‐derived cells, potentially via increased TMEM16A (Ano1). Such increases may further contribute to the observed hormone‐induced enhancement of fibroid cell proliferation. Menstrual cycle fluctuations in reproductive aged women may thus contribute to the progression of fibroids. Greater understanding of calcium signaling pathways such as Ano1 in fibroid tissue may allow for novel therapeutic targets in uterine leiomyomas.Support or Funding InformationSupported by NIH grants R25 GMO75148 (JLR), R01 HL088029 (YSP), and R01 HL56470 (YSP).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium Spark Activity is Modulated by Perfusion Pressure in Vascular Smooth Muscle of Afferent Arterioles

Myogenic response and tubuloglomerular feedback are the two primary mechanisms for renal blood flow autoregulation by regulating the afferent arteriolar vascular resistance. However, the mechanisms of mechanotransduction in pressure induced myogenic response are not well defined in afferent arterioles. We have previously demonstrated that integrin‐mediated mechanical force increases the occurrence of Ca2+ sparks in freshly isolated renal vascular smooth muscle cells (VSMCs). To test whether generation of Ca2+ sparks is a downstream signal of mechanotransduction in pressure induced myogenic constriction, the relationship between Ca2+ spark frequency and transmural perfusion pressure was investigated in intact VSMCs of afferent arterioles isolated from Sprague‐Dawley rats. Spontaneous Ca2+ sparks were found in VSMCs when afferent arterioles were perfused at 80 mmHg. The Ca2+ spark frequency was significantly increased when perfusion pressure was increased by 40 mmHg. Similar increase of Ca2+ spark frequency was also found when arterioles were stimulated with β1‐integrin activating antibody. Spontaneous membrane current recorded with whole cell perforated‐patch in renal VSMCs indicated that there were more spontaneous transient inward currents (STICs) than spontaneous transient outward currents (STOCs) when holding potential was set close to physiological resting membrane potential. These observations indicated that Ca2+ sparks activity might couple to Ca2+‐activated Cl− (ClCa) channel and trigger pressure induced myogenic constriction via membrane depolarization. Real‐time PCR and immunofluorescence confirmed the presence of the ClCa channel TMEM16A in renal VSMCs. Inhibition of TMEM16A with the specific antagonist T16Ainh‐A01 impaired pressure induced myogenic contraction in the perfused afferent arterioles. It is known that myogenic response is enhanced in spontaneously hypertensive rat (SHR). TMEM16A abundance and spontaneous Ca2+ sparks were more prevalent in renal arterioles of SHR than those of Sprague‐Dawley rats, which are consistent with notion that Ca2+ spark activity is part of the signal transduction process in myogenic response. In conclusion, Ca2+ sparks were detected in intact VSMCs of afferent arterioles for the first time and their frequencies were positively modulated by the perfusion pressure. The coupling Ca2+ sparks to ClCa might contribute to the mechanical signal transduction process in pressure induced myogenic constriction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of Anion Conduction in the Calcium-Activated Chloride Channel TMEM16A

The calcium-activated chloride channel TMEM16A mediates selective anion conduction upon activation by intracellular calcium. Using the cryo-EM structure of mouse TMEM16A as a template, we have investigated the mechanism of anion conduction by patch-clamp electrophysiology. In the structure, an hourglass-shaped, protein-enclosed aqueous conduit populated by basic residues is found in each subunit in a homodimer. We analysed the conduction characteristics of alanine mutants of these basic residues and found a position-dependent effect on ion conduction that is manifested in the rectification of current-voltage relationships. This underlines their importance for the electrostatics in the narrow neck region of the pore creating a favourable environment for anion conduction. The compromised conduction characteristics of the cysteine mutant of a basic residue located on α-helix 5 at the intracellular end of the narrow neck can be reverted to the wild-type phenotype upon reaction with the small, positively charged MTSEA, indicating the intracellular accessibility of the site and reinforcing the role of the positive potential for anion conduction. The equivalent mutation of a residue located one helix turn towards the extracellular side is inaccessible, consistent with its location in the constricted part of the pore. When analysed in the context of a phenomenological model of ion permeation, it appears that the positively charged residues in the aqueous pore facilitate anion conduction by lowering local energy barrier(s) according to their specific position in the pore. Because the major energy barriers are located at the inner and outer ends of the pore, it is plausible that anions dehydrate as they enter the narrow conduit for conduction, a process that might be facilitated by the resident basic residues.

PIP2 and CA2+ are Both Required to Open TMEM16a Channels in Xenopus Laevis Oocytes

PIP2 and CA2+ are Both Required to Open TMEM16a Channels in Xenopus Laevis Oocytes

TMEM16A and TMEM16B channel proteins generate Ca2+-activated Cl− current and regulate melatonin secretion in rat pineal glands

Pinealocytes regulate circadian rhythm by synthesizing and secreting melatonin. These cells generate action potentials; however, the contribution of specific ion channels to melatonin secretion from pinealocytes remains unclear. In this study, the involvement and molecular identity of Ca2+-activated Cl- (ClCa) channels in the regulation of melatonin secretion were examined in rat pineal glands. Treatment with the ClCa channel blockers, niflumic acid or T16Ainh-A01, significantly reduced melatonin secretion in pineal glands. After pineal K+ currents were totally blocked under whole-cell patch clamp conditions, depolarization and subsequent repolarization induced a slowly activating outward current and a substantial inward tail current, respectively. Both of these current changes were dependent on intracellular Ca2+ concentration and inhibited by niflumic acid and T16Ainh-A01. Quantitative real-time PCR, Western blotting, and immunocytochemical analyses revealed that TMEM16A and TMEM16B were highly expressed in pineal glands. siRNA knockdown of TMEM16A and/or TMEM16B showed that both channels contribute to ClCa currents in pinealocytes. Conversely, co-expression of TMEM16A and TMEM16B channels or the expression of this tandem channel in HEK293 cells mimicked the electrophysiological characteristics of ClCa currents in pinealocytes. Moreover, bimolecular fluorescence complementation, FRET, and co-immunoprecipitation experiments suggested that TMEM16A and TMEM16B can form heteromeric channels, as well as homomeric channels. In conclusion, pineal ClCa channels are composed of TMEM16A and TMEM16B subunits, and these fluxes regulate melatonin secretion in pineal glands.

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1)

The TMEM16A-mediated Ca2+-activated Cl− current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.

Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.

The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca2+ concentration. The protein is broadly expressed and contributes to diverse physiological processes, including transepithelial chloride transport and the control of electrical signalling in smooth muscles and certain neurons. As a member of the TMEM16 (or anoctamin) family of membrane proteins, TMEM16A is closely related to paralogues that function as scramblases, which facilitate the bidirectional movement of lipids across membranes. The unusual functional diversity of the TMEM16 family and the relationship between two seemingly incompatible transport mechanisms has been the focus of recent investigations. Previous breakthroughs were obtained from the X-ray structure of the lipid scramblase of the fungus Nectria haematococca (nhTMEM16), and from the cryo-electron microscopy structure of mouse TMEM16A at 6.6 Å (ref. 14). Although the latter structure disclosed the architectural differences that distinguish ion channels from lipid scramblases, its low resolution did not permit a detailed molecular description of the protein or provide any insight into its activation by Ca2+. Here we describe the structures of mouse TMEM16A at high resolution in the presence and absence of Ca2+. These structures reveal the differences between ligand-bound and ligand-free states of a calcium-activated chloride channel, and when combined with functional experiments suggest a mechanism for gating. During activation, the binding of Ca2+ to a site located within the transmembrane domain, in the vicinity of the pore, alters the electrostatic properties of the ion conduction path and triggers a conformational rearrangement of an α-helix that comes into physical contact with the bound ligand, and thereby directly couples ligand binding and pore opening. Our study describes a process that is unique among channel proteins, but one that is presumably general for both functional branches of the TMEM16 family.

Identification of Resveratrol, an Herbal Compound, as an Activator of the Calcium-Activated Chloride Channel, TMEM16A

Calcium-activated chloride channels (CaCCs) play vital roles in a variety of physiological processes. Dysfunction of the CaCCs is implicated in many diseases. Drug discovery targeting at CaCCs has recently become possible with the determination that TMEM16A is the molecular identity of CaCCs. In this study, we demonstrated that resveratrol (RES), a Chinese traditional medicine compound, is a novel activator of TMEM16A. The yellow fluorescence protein quenching assay and measurement of intracellular calcium fluorescence intensity show that RES activates TMEM16A channels in an intracellular Ca2+-independent way. The data of inside-out patch clamp revealed that RES dose-dependently activates TMEM16A (EC50=47.92±9.35μM). Furthermore, RES enhanced the contractions of the ileum of guinea pigs by activating the TMEM16A channel, which indicated that RES might be a promising drug for the treatment of gastrointestinal hypomotility. As RES was able to induce TMEM16A channel activation, TMEM16A can be added to the list of RES drug targets.