Predicting Mutational Effects on Ca2+-Activated Chloride Conduction of TMEM16A Based on a Simulation Study.

The dysfunction and defects of ion channels are associated with many human diseases, especially for loss-of-function mutations in ion channels such as cystic fibrosis transmembrane conductance regulator mutations in cystic fibrosis. Understanding ion channels is of great current importance for both medical and fundamental purposes. Such an understanding should include the ability to predict mutational effects and describe functional and mechanistic effects. In this work, we introduce an approach to predict mutational effects based on kinetic information (including reaction barriers and transition state locations) obtained by studying the working mechanism of target proteins. Specifically, we take the Ca2+-activated chloride channel TMEM16A as an example and utilize the computational biology model to predict the mutational effects of key residues. Encouragingly, we verified our predictions through electrophysiological experiments, demonstrating a 94% prediction accuracy regarding mutational directions. The mutational strength assessed by Pearson's correlation coefficient is -0.80 between our calculations and the experimental results. These findings suggest that the proposed methodology is reliable and can provide valuable guidance for revealing functional mechanisms and identifying key residues of the TMEM16A channel. The proposed approach can be extended to a broad scope of biophysical systems.

The role of Transmembrane Protein 16A (TMEM16A) in pulmonary hypertension.

Transmembrane protein 16A (TMEM16A), a member of the TMEM16 family, is the molecular basis of Ca2+-activated chloride channels (CaCCs) and is involved in a variety of physiological and pathological processes. Previous studies have focused more on respiratory-related diseases and tumors. However, recent studies have identified an important role for TMEM16A in cardiovascular diseases, especially in pulmonary hypertension. TMEM16A is expressed in both pulmonary artery smooth muscle cells and pulmonary artery endothelial cells and is involved in the development of pulmonary hypertension. This paper presents the structure and function of TMEM16A, the pathogenesis of pulmonary hypertension, and highlights the role and mechanism of TMEM16A in pulmonary hypertension, summarizing the controversies in this field and taking into account hypertension and portal hypertension, which have similar pathogenesis. It is hoped that the unique role of TMEM16A in pulmonary hypertension will be illustrated and provide ideas for research in this area.

Gating and anion selectivity are reciprocally regulated in TMEM16A (ANO1).

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.

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.

Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization.

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions.

The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1-3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane.

ClC-3 promotes paclitaxel resistance via modulating tubulins polymerization in ovarian cancer cells.

Epithelial ovarian cancers (EOC) present as malignant tumors with high mortality in the female reproductive system diseases. Acquired resistance to paclitaxel (PTX), one of the first-line treatment of EOC, remains a therapeutic challenge. ClC-3, a member of the voltage-gated Cl- channels, plays an essential role in a variety of cellular activities, including chemotherapeutic resistance. Here, we demonstrated that the protein expression and channel function of ClC-3 was upregulated in PTX resistance A2780/PTX cells compared with its parental A2780 cells. The silence of ClC-3 expression by siRNA in A2780/PTX cells partly recovered the PTX sensitivity through restored the G2/M arrest and resumed the chloride channel blocked. ClC-3 siRNA both inhibited the expression of ClC-3 and ß-tubulin, whereas the ß-tubulin siRNA reduced the expression of itself only, without affecting the expression of ClC-3. Moreover, treatment of ClC-3 siRNA in A2780/PTX cells increased the polymerization ratio of ß-tubulin, and the possibility of proteins interaction between ClC-3 and ß-tubulin was existing. Take together, the over-expression of ClC-3 protein in PTX-resistance ovarian cancer cells promotes the combination of ClC-3 and ß-tubulin, which in turn increase the ration of free form and decrease the quota of the polymeric form of ß-tubulin, and finally reduce the sensitivity to PTX. Our findings elucidated a novel function of ClC-3 in regulating PTX resistance and ClC-3 could serve as a potential target to overcome the PTX resistance ovarian cancer.

Conserved cysteine dioxidation enhances membrane interaction of human Cl- intracellular channel 5.

The human chloride intracellular channel (hCLIC) family is thought to transition between globular and membrane-associated forms by exposure of a hydrophobic surface. However, the molecular identity of this surface, and the triggering events leading to its exposure, remain elusive. Here, by combining biochemical and structural approaches, together with mass spectrometry (MS) analyses, we show that hCLIC5 is inherently flexible. X-ray crystallography revealed the existence of a globular conformation, while small-angle X-ray scattering showed additional elongated forms consisting of exposure of the conserved hydrophobic inter-domain interface to the bulk phase. Tryptophan fluorescence measurements demonstrated that the transition to the membrane-associated form is enhanced by the presence of oxidative environment and lipids. Using MS, we identified a dose-dependent oxidation of a highly conserved cysteine residue, known to play a key role in the structurally related omega-class of glutathione-S-transferases. Hydrogen/deuterium exchange MS analysis revealed that oxidation of this cysteine facilitates the exposure of the conserved hydrophobic inter-domain interface. Together, our results pinpoint an oxidation of a specific cysteine residue as a triggering mechanism initializing the molecular commitment for membrane interaction in the CLIC family.

Isolation and characterization of a novel member of the ACC ligand-gated chloride channel family, Hco-LCG-46, from the parasitic nematode Haemonchus contortus.

The ACC-1 family of cys-loop receptors are ligand-gated chloride channels sensitive to acetylcholine (ACh), and are only present in invertebrates. Studies of this family of inhibitory receptors has provided insight into how they bind and respond to ACh in a manner vastly different from nicotinic acetylcholine receptors and appear to be present in tissues that are relevant to anthelmintic action. Here, we have identified two members of the ACC-1 family from the parasitic nematode Haemonchus contortus, Hco-LGC-46 and Hco-ACC-4. Hco-LGC-46 is an ACC subunit that has never been previously expressed and pharmacologically characterized. We found that Hco-LGC-46 when expressed in Xenopus laevis oocytes forms a functional homomeric channel that is responsive to the cholinergic agonists ACh and methylcholine. hco-lgc-46 expressed in a C. elegans lgc-46 null strain (ok2900) suppressed hypersensitivity to aldicarb in a manner similar to cel-lgc-46. It was also found that Hco-LGC-46 assembles with Hco-ACC-1 and produces a receptor that is over 5-fold more sensitive to ACh and responds to the cholinergic agonists methycholine and carbachol. In contrast, the co-expression of Hco-LGC-46 with Hco-ACC-4 resulted in non-functional channels in oocytes. Hco-ACC-4 also appears to form heteromeric channels with a previously characterized subunit, Hco-ACC-2. Co-expression of Hco-ACC-4 with Hco-ACC-2 resulted in a functional heteromeric channel with an EC50 value similar to that of the Hco-ACC-2 homomeric channel. However, the maximum currents generated in the ACC-4/ACC-2 channel were significantly (p < 0.005) lower than those from the ACC-2 homomeric channel. Overall, this is the first report confirming that lgc-46 encodes an acetylcholine-gated chloride channel which when co-expressed with acc-4 results in reduced receptor function or trafficking in oocytes.

Structural and Biophysical Analysis of the CLCA1 VWA Domain Suggests Mode of TMEM16A Engagement.

The secreted protein calcium-activated chloride channel regulator 1 (CLCA1) utilizes a von Willebrand factor type A (VWA) domain to bind to and potentiate the calcium-activated chloride channel TMEM16A. To gain insight into this unique potentiation mechanism, we determined the 2.0-Å crystal structure of human CLCA1 VWA bound to Ca2+. The structure reveals the metal-ion-dependent adhesion site (MIDAS) in a high-affinity "open" conformation, engaging in crystal contacts that likely mimic how CLCA1 engages TMEM16A. The CLCA1 VWA contains a disulfide bond between α3 and α4 in close proximity to the MIDAS that is invariant in the CLCA family and unique in VWA structures. Further biophysical studies indicate that CLCA1 VWA is preferably stabilized by Mg2+ over Ca2+ and that α6 atypically extends from the VWA core. Finally, an analysis of TMEM16A structures suggests residues likely to mediate interaction with CLCA1 VWA.

Intracellular Ion Channels in Pancreas Cancer.

The current basis of systemic treatment for pancreas cancer involves cytotoxic chemotherapy. Despite newer regimens, overall survival remains poor and this dilemma is further compounded by a lack of effective, novel therapeutic targets. Another challenge in treating pancreas cancer is the complex tumor microenvironment, which contains regulatory T cells, myeloid derived suppressor cells, and tumor associated macrophages that forms a barrier to standard therapies. Intracellular ion channels are ubiquitously expressed in all cells and their role in carcinogenesis is increasingly becoming elucidated. They play an integral role in each of the six "Hallmarks of Cancer" and are potential novel prognostic biomarkers and therapeutic targets for pancreas cancer. Although examined in various hematologic and gastrointestinal malignancies, there are limited data examining the prognostic role of specific ion channels in pancreas ductal adenocarcinoma. This review focuses on chloride (CLCA-1, CLIC1, CLIC3), calcium (TRPM7, TRPM8), and potassium (Kir3.1, KCa3.1, Kv11.1, Kv1.3) channels in pancreas cancer.

Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2-processing properties.

Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.

Molecular identification of HSPA8 as an accessory protein of a hyperpolarization-activated chloride channel from rat pulmonary vein cardiomyocytes.

Pulmonary veins (PVs) are the major origin of atrial fibrillation. Recently, we recorded hyperpolarization-activated Cl- current (ICl, h) in rat PV cardiomyocytes. Unlike the well-known chloride channel protein 2 (CLCN2) current, the activation curve of ICl, h was hyperpolarized as the Cl- ion concentration ([Cl-] i ) increased. This current could account for spontaneous activity in PV cardiomyocytes linked to atrial fibrillation. In this study, we aimed to identify the channel underlying ICl, h Using RT-PCR amplification specific for Clcn2 or its homologs, a chloride channel was cloned from rat PV and detected in rat PV cardiomyocytes using immunocytochemistry. The gene sequence and electrophysiological functions of the protein were identical to those previously reported for Clcn2, with protein activity observed as a hyperpolarization-activated current by the patch-clamp method. However, the [Cl-] i dependence of activation was entirely different from the observed ICl, h of PV cardiomyocytes; the activation curve of the Clcn2-transfected cells shifted toward positive potential with increased [Cl-] i , whereas the ICl, h of PV and left ventricular cardiomyocytes showed a leftward shift. Therefore, we used MS to explore the possibility of additional proteins interacting with CLCN2 and identified an individual 71-kDa protein, HSPA8, that was strongly expressed in rat PV cardiomyocytes. With co-expression of HSPA8 in HEK293 and PC12 cells, the CLCN2 current showed voltage-dependent activation and shifted to negative potential with increasing [Cl-] i Molecular docking simulations further support an interaction between CLCN2 and HSPA8. These findings suggest that CLCN2 in rat heart contains HSPA8 as a unique accessory protein.

Structure of the human ClC-1 chloride channel.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-ß-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.

A G-Quadruplex Structure in the Promoter Region of CLIC4 Functions as a Regulatory Element for Gene Expression.

The differential transcriptional expression of CLIC4 between tumor cells and the surrounding stroma during cancer progression has been suggested to have a tumor-promoting effect. However, little is known about the transcriptional regulation of CLIC4. To better understand how this gene is regulated, the promoter region of CLIC4 was analyzed. We found that a high GC content near the transcriptional start site (TSS) might form an alternative G-quadruplex (G4) structure. Nuclear magnetic resonance spectroscopy (NMR) confirmed their formation in vitro. The reporter assay showed that one of the G4 structures exerted a regulatory role in gene transcription. When the G4-forming sequence was mutated to disrupt the G4 structure, the transcription activity dropped. To examine whether this G4 structure actually has an influence on gene transcription in the chromosome, we utilized the CRISPR/Cas9 system to edit the G4-forming sequence within the CLIC4 promoter in the cell genome. The pop-in/pop-out strategy was adopted to isolate the precisely-edited A375 cell clone. In CRISPR-modified A375 cell clones whose G4 was disrupted, there was a decrease in the endogenous CLIC4 messenger RNA (mRNA) expression level. In conclusion, we found that the G4 structure in the CLIC4 promoter might play an important role in regulating the level of transcription.

Calcium-Activated Cl- Channel: Insights on the Molecular Identity in Epithelial Tissues.

Calcium-activated chloride secretion in epithelial tissues has been described for many years. However, the molecular identity of the channel responsible for the Ca2+-activated Cl− secretion in epithelial tissues has remained a mystery. More recently, TMEM16A has been identified as a new putative Ca2+-activated Cl− channel (CaCC). The primary goal of this article will be to review the characterization of TMEM16A, as it relates to the physical structure of the channel, as well as important residues that confer voltage and Ca2+-sensitivity of the channel. This review will also discuss the role of TMEM16A in epithelial physiology and potential associated-pathophysiology. This will include discussion of developed knockout models that have provided much needed insight on the functional localization of TMEM16A in several epithelial tissues. Finally, this review will examine the implications of the identification of TMEM16A as it pertains to potential novel therapies in several pathologies.

Molecular, biophysical, and pharmacological properties of calcium-activated chloride channels.

Calcium-activated chloride channels (CaCCs) are a family of anionic transmembrane ion channels. They are mainly responsible for the movement of Cl- and other anions across the biological membranes, and they are widely expressed in different tissues. Since the Cl- flow into or out of the cell plays a crucial role in hyperpolarizing or depolarizing the cells, respectively, the impact of intracellular Ca2+ concentration on these channels is attracting a lot of attentions. After summarizing the molecular, biophysical, and pharmacological properties of CaCCs, the role of CaCCs in normal cellular functions will be discussed, and I will emphasize how dysregulation of CaCCs in pathological conditions can account for different diseases. A better understanding of CaCCs and a pivotal regulatory role of Ca2+ can shed more light on the therapeutic strategies for different neurological disorders that arise from chloride dysregulation, such as asthma, cystic fibrosis, and neuropathic pain.

Identification of Potent Chloride Intracellular Channel Protein 1 Inhibitors from Traditional Chinese Medicine through Structure-Based Virtual Screening and Molecular Dynamics Analysis.

Chloride intracellular channel 1 (CLIC1) is involved in the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has become an attractive new therapeutic target for several types of cancer. In this work, we aim to identify natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM) database using structure-based virtual screening and molecular dynamics (MD) simulation. First, structure-based docking was employed to screen the refined TCM database and the top 500 TCM compounds were obtained and reranked by X-Score. Then, 30 potent hits were achieved from the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) analysis was used to refine the virtual hits. Six TCM compounds with top MM-GBSA scores and ideal-binding models were confirmed as the final hits. Our study provides information about the interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In addition, the top 6 natural products structural scaffolds could serve as building blocks in designing drug-like molecules for CLIC1 inhibition.

Cationic liquid crystalline nanoparticles for the delivery of synthetic RNAi-based therapeutics.

RNA interference (RNAi)-based therapeutics have been used to silence the expression of targeted pathological genes. Small interfering RNA (siRNAs) and microRNA (miRNAs) inhibitor have performed this function. However, short half-life, poor cellular uptake, and nonspecific distribution of small RNAs call for the development of novel delivery systems to facilitate the use of RNAi. We developed a novel cationic liquid crystalline nanoparticle (CLCN) to efficiently deliver synthetic siRNAs and miRNAs. CLCNs were prepared by using high-speed homogenization and assembled with synthetic siRNA or miRNA molecules in nuclease-free water to create CLCN/siRNA or miRNA complexes. The homogeneous and stable CLCNs and CLCN-siRNA complexes were about 100 nm in diameter, with positively charged surfaces. CLCNs are nontoxic and are taken up by human cells though endocytosis. Significant inhibition of gene expression was detected in transiently transfected lung cancer H1299 cells treated with CLCNs/anti-GFP complexes 24 hours after transfection. Biodistribution analysis showed that the CLCNs and CLCNs-RNAi complexes were successfully delivered to various organs and into the subcutaneous human lung cancer H1299 tumor xenografts in mice 24 hours after systemic administration. These results suggest that CLCNs are a unique and advanced delivery system capable of protecting RNAi from degradation and of efficiently delivering RNAi in vitro and in vivo.

Research and progress on ClC­2 (Review).

Chloride channel 2 (ClC-2) is one of the nine mammalian members of the ClC family. The present review discusses the molecular properties of ClC­2, including CLCN2, ClC­2 promoter and the structural properties of ClC­2 protein; physiological properties; functional properties, including the regulation of cell volume. The effects of ClC­2 on the digestive, respiratory, circulatory, nervous and optical systems are also discussed, in addition to the mechanisms involved in the regulation of ClC­2. The review then discusses the diseases associated with ClC­2, including degeneration of the retina, Sjögren's syndrome, age­related cataracts, degeneration of the testes, azoospermia, lung cancer, constipation, repair of impaired intestinal mucosa barrier, leukemia, cystic fibrosis, leukoencephalopathy, epilepsy and diabetes mellitus. It was concluded that future investigations of ClC­2 are likely to be focused on developing specific drugs, activators and inhibitors regulating the expression of ClC­2 to treat diseases associated with ClC­2. The determination of CLCN2 is required to prevent and treat several diseases associated with ClC­2.