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Cells Oct 2021Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in a gene encoding a protein called Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).... (Review)
Review
Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in a gene encoding a protein called Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The CFTR protein is known to acts as a chloride (Cl) channel expressed in the exocrine glands of several body systems where it also regulates other ion channels, including the epithelial sodium (Na) channel (ENaC) that plays a key role in salt absorption. This function is crucial to the osmotic balance of the mucus and its viscosity. However, the pathophysiology of CF is more challenging than a mere dysregulation of epithelial ion transport, mainly resulting in impaired mucociliary clearance (MCC) with consecutive bronchiectasis and in exocrine pancreatic insufficiency. This review shows that the CFTR protein is not just a chloride channel. For a long time, research in CF has focused on abnormal Cl and Na transport. Yet, the CFTR protein also regulates numerous other pathways, such as the transport of HCO, glutathione and thiocyanate, immune cells, and the metabolism of lipids. It influences the pH homeostasis of airway surface liquid and thus the MCC as well as innate immunity leading to chronic infection and inflammation, all of which are considered as key pathophysiological characteristics of CF.
Topics: Animals; Chloride Channels; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelium; Humans; Lipid Metabolism; Models, Biological
PubMed: 34831067
DOI: 10.3390/cells10112844 -
Physiological Reviews Apr 2014TMEM16 proteins, also known as anoctamins, are involved in a variety of functions that include ion transport, phospholipid scrambling, and regulation of other membrane... (Review)
Review
TMEM16 proteins, also known as anoctamins, are involved in a variety of functions that include ion transport, phospholipid scrambling, and regulation of other membrane proteins. The first two members of the family, TMEM16A (anoctamin-1, ANO1) and TMEM16B (anoctamin-2, ANO2), function as Ca2+-activated Cl- channels (CaCCs), a type of ion channel that plays important functions such as transepithelial ion transport, smooth muscle contraction, olfaction, phototransduction, nociception, and control of neuronal excitability. Genetic ablation of TMEM16A in mice causes impairment of epithelial Cl- secretion, tracheal abnormalities, and block of gastrointestinal peristalsis. TMEM16A is directly regulated by cytosolic Ca2+ as well as indirectly by its interaction with calmodulin. Other members of the anoctamin family, such as TMEM16C, TMEM16D, TMEM16F, TMEM16G, and TMEM16J, may work as phospholipid scramblases and/or ion channels. In particular, TMEM16F (ANO6) is a major contributor to the process of phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane. Intriguingly, TMEM16F is also associated with the appearance of anion/cation channels activated by very high Ca2+ concentrations. Furthermore, a TMEM16 protein expressed in Aspergillus fumigatus displays both ion channel and lipid scramblase activity. This finding suggests that dual function is an ancestral characteristic of TMEM16 proteins and that some members, such as TMEM16A and TMEM16B, have evolved to a pure channel function. Mutations in anoctamin genes (ANO3, ANO5, ANO6, and ANO10) cause various genetic diseases. These diseases suggest the involvement of anoctamins in a variety of cell functions whose link with ion transport and/or lipid scrambling needs to be clarified.
Topics: Animals; Chloride Channels; Disease Models, Animal; Genetic Predisposition to Disease; Humans; Membrane Transport Modulators; Mutation; Protein Conformation; Signal Transduction; Structure-Activity Relationship
PubMed: 24692353
DOI: 10.1152/physrev.00039.2011 -
American Journal of Physiology. Cell... Dec 2021Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and... (Review)
Review
Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride-transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers, and cotransporters, some having broad anion specificity. Loss-of-function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt-wasting nephropathy, myotonia, osteopetrosis, hearing loss, and goiter. Although impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for the development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension, and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.
Topics: Animals; Antiporters; Chloride Channels; Chlorides; Cystic Fibrosis Transmembrane Conductance Regulator; Drug Design; Drug Discovery; Humans; Ion Transport; Kinetics; Membrane Transport Modulators; Mutation; Sulfate Transporters
PubMed: 34644122
DOI: 10.1152/ajpcell.00334.2021 -
Proceedings of the National Academy of... Aug 2022In response to acidic pH, the widely expressed proton-activated chloride (PAC) channel opens and conducts anions across cellular membranes. By doing so, PAC plays an...
In response to acidic pH, the widely expressed proton-activated chloride (PAC) channel opens and conducts anions across cellular membranes. By doing so, PAC plays an important role in both cellular physiology (endosome acidification) and diseases associated with tissue acidosis (acid-induced cell death). Despite the available structural information, how proton binding in the extracellular domain (ECD) leads to PAC channel opening remains largely unknown. Here, through comprehensive mutagenesis and electrophysiological studies, we identified several critical titratable residues, including two histidine residues (H130 and H131) and an aspartic acid residue (D269) at the distal end of the ECD, together with the previously characterized H98 at the transmembrane domain-ECD interface, as potential pH sensors for human PAC. Mutations of these residues resulted in significant changes in pH sensitivity. Some combined mutants also exhibited large basal PAC channel activities at neutral pH. By combining molecular dynamics simulations with structural and functional analysis, we further found that the β12 strand at the intersubunit interface and the associated "joint region" connecting the upper and lower ECDs allosterically regulate the proton-dependent PAC activation. Our studies suggest a distinct pH-sensing and gating mechanism of this new family of ion channels sensitive to acidic environment.
Topics: Aspartic Acid; Chloride Channels; Chlorides; Electrophysiological Phenomena; Histidine; Humans; Hydrogen-Ion Concentration; Mutagenesis; Protons
PubMed: 35878032
DOI: 10.1073/pnas.2200727119 -
International Journal of Molecular... Jan 2022The TMEM16A/anoctamin-1 calcium-activated chloride channel (CaCC) contributes to a range of vital functions, such as the control of vascular tone and epithelial ion... (Review)
Review
The TMEM16A/anoctamin-1 calcium-activated chloride channel (CaCC) contributes to a range of vital functions, such as the control of vascular tone and epithelial ion transport. The channel is a founding member of a family of 10 proteins (TMEM16x) with varied functions; some members (i.e., TMEM16A and TMEM16B) serve as CaCCs, while others are lipid scramblases, combine channel and scramblase function, or perform additional cellular roles. TMEM16x proteins are typically activated by agonist-induced Ca release evoked by G-protein-coupled receptor (GPCR) activation; thus, TMEM16x proteins link Ca-signalling with cell electrical activity and/or lipid transport. Recent studies demonstrate that a range of other cellular factors-including plasmalemmal lipids, pH, hypoxia, ATP and auxiliary proteins-also control the activity of the TMEM16A channel and its paralogues, suggesting that the TMEM16x proteins are effectively polymodal sensors of cellular homeostasis. Here, we review the molecular pathophysiology, structural biology, and mechanisms of regulation of TMEM16x proteins by multiple cellular factors.
Topics: Animals; Anoctamin-1; Anoctamins; Biological Transport; Cell Membrane; Chloride Channels; Humans; Ion Transport; Phospholipid Transfer Proteins
PubMed: 35163502
DOI: 10.3390/ijms23031580 -
ELife Dec 2022Desensitization is a common property of membrane receptors, including ion channels. The newly identified proton-activated chloride (PAC) channel plays an important role...
Desensitization is a common property of membrane receptors, including ion channels. The newly identified proton-activated chloride (PAC) channel plays an important role in regulating the pH and size of organelles in the endocytic pathway, and is also involved in acid-induced cell death. However, how the PAC channel desensitizes is largely unknown. Here, we show by patch-clamp electrophysiological studies that PAC (also known as TMEM206/ASOR) undergoes pH-dependent desensitization upon prolonged acid exposure. Through structure-guided and comprehensive mutagenesis, we identified several residues critical for PAC desensitization, including histidine (H) 98, glutamic acid (E) 94, and aspartic acid (D) 91 at the extracellular extension of the transmembrane helix 1 (TM1), as well as E107, D109, and E250 at the extracellular domain (ECD)-transmembrane domain (TMD) interface. Structural analysis and molecular dynamic simulations revealed extensive interactions between residues at the TM1 extension and those at the ECD-TMD interface. These interactions likely facilitate PAC desensitization by stabilizing the desensitized conformation of TM1, which undergoes a characteristic rotational movement from the resting and activated states to the desensitized state. Our studies establish a new paradigm of channel desensitization in this ubiquitously expressed ion channel and pave the way for future investigation of its relevance in cellular physiology and disease.
Topics: Protons; Chlorides; Chloride Channels; Protein Domains
PubMed: 36547405
DOI: 10.7554/eLife.82955 -
Molecular Medicine Reports Jul 2017Chloride channel 2 (ClC-2) is one of the nine mammalian members of the ClC family. The present review discusses the molecular properties of ClC‑2, including CLCN2,... (Review)
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 ClC‑2, including CLCN2, ClC‑2 promoter and the structural properties of ClC‑2 protein; physiological properties; functional properties, including the regulation of cell volume. The effects of ClC‑2 on the digestive, respiratory, circulatory, nervous and optical systems are also discussed, in addition to the mechanisms involved in the regulation of ClC‑2. The review then discusses the diseases associated with ClC‑2, including degeneration of the retina, Sjögren's syndrome, age‑related 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 ClC‑2 are likely to be focused on developing specific drugs, activators and inhibitors regulating the expression of ClC‑2 to treat diseases associated with ClC‑2. The determination of CLCN2 is required to prevent and treat several diseases associated with ClC‑2.
Topics: Animals; CLC-2 Chloride Channels; Carrier Proteins; Chloride Channels; Disease Susceptibility; Gene Expression Regulation; Humans; Protein Binding; Research; Structure-Activity Relationship
PubMed: 28534947
DOI: 10.3892/mmr.2017.6600 -
Channels (Austin, Tex.) 2016Ca(2+)-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle... (Review)
Review
Ca(2+)-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca(2+) sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca(2+)-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca(2+)-dependent sensitization of activation (CDSA) and Ca(2+)-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca(2+)-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.
Topics: Alternative Splicing; Animals; Binding Sites; Calcium; Calmodulin; Chloride Channels; Humans; Ion Channel Gating
PubMed: 26083059
DOI: 10.1080/19336950.2015.1058455 -
Journal of Biomedicine & Biotechnology 2011The CLC-1 chloride channel, a member of the CLC-channel/transporter family, plays important roles for the physiological functions of skeletal muscles. The opening of... (Review)
Review
The CLC-1 chloride channel, a member of the CLC-channel/transporter family, plays important roles for the physiological functions of skeletal muscles. The opening of this chloride channel is voltage dependent and is also regulated by protons and chloride ions. Mutations of the gene encoding CLC-1 result in a genetic disease, myotonia congenita, which can be inherited as an autosmal dominant (Thomsen type) or an autosomal recessive (Becker type) pattern. These mutations are scattered throughout the entire protein sequence, and no clear relationship exists between the inheritance pattern of the mutation and the location of the mutation in the channel protein. The inheritance pattern of some but not all myotonia mutants can be explained by a working hypothesis that these mutations may exert a "dominant negative" effect on the gating function of the channel. However, other mutations may be due to different pathophysiological mechanisms, such as the defect of protein trafficking to membranes. Thus, the underlying mechanisms of myotonia are likely to be quite diverse, and elucidating the pathophysiology of myotonia mutations will require the understanding of multiple molecular/cellular mechanisms of CLC-1 channels in skeletal muscles, including molecular operation, protein synthesis, and membrane trafficking mechanisms.
Topics: Animals; Chloride Channels; Female; Humans; Male; Models, Molecular; Mutation; Myotonia Congenita
PubMed: 22187529
DOI: 10.1155/2011/685328 -
The Journal of Biological Chemistry Nov 2023Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is...
Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K) solutions, we determined that CLIC6 is more permeable to chloride-(Cl) as compared to bromide-(Br), fluoride-(F), and K ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl channel.
Topics: Animals; Humans; Mice; Anions; Chloride Channels; Chlorides; Epithelial Cells; HEK293 Cells
PubMed: 37838179
DOI: 10.1016/j.jbc.2023.105349