TMEM16 proteins, also known as anoctamins, are a family of ten membrane proteins with various tissue expression and subcellular localization. TMEM16A (anoctamin 1) is a plasma membrane protein that acts as a calcium-activated chloride channel. It is expressed in many types of epithelial cells, smooth muscle cells and some neurons. In airway epithelial cells, TMEM16A expression is particularly enhanced by inflammatory stimuli that also promote goblet cell metaplasia and mucus hypersecretion. Therefore, pharmacological modulation of TMEM16A could be beneficial to improve mucociliary clearance in chronic obstructive respiratory diseases. However, the correct approach to modulate TMEM16A activity (activation or inhibition) is still debated. Pharmacological inhibitors of TMEM16A could also be useful as anti-hypertensive agents given the TMEM16A role in smooth muscle contraction. In contrast to TMEM16A, TMEM16F (anoctamin 6) behaves as a calcium-activated phospholipid scramblase, responsible for the externalization of phosphatidylserine on cell surface. Inhibitors of TMEM16F could be useful as anti-coagulants and anti-viral agents. The role of other anoctamins as therapeutic targets is still unclear since their physiological role is still to be defined.

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.

The cGAS-STING signaling pathway has emerged as a key mediator of inflammation. However, the roles of chloride homeostasis on this pathway are unclear. Here, we uncovered a correlation between chloride homeostasis and cGAS-STING signaling. We found that dysregulation of chloride homeostasis attenuates cGAS-STING signaling in a lysosome-independent manner. Treating immune cells with chloride channel inhibitors attenuated 2\'3\'-cGAMP production by cGAS and also suppressed STING polymerization, leading to reduced cytokine production. We also demonstrate that non-selective chloride channel blockers can suppress the NPC1 deficiency-induced, hyper-activated STING signaling in skin fibroblasts derived from Niemann Pick disease type C (NPC) patients. Our findings reveal that chloride homeostasis majorly affects cGAS-STING pathway and suggest a provocative strategy to dampen STING-mediated inflammation via targeting chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates transepithelial salt and fluid homeostasis. CFTR dysfunction leads to reduced chloride secretion into the mucosal lining of epithelial tissues, thereby causing the inherited disease cystic fibrosis. Although several structures of CFTR are available, our understanding of the ion-conduction pathway is incomplete. In particular, the route that connects the cytosolic vestibule with the extracellular space has not been clearly defined, and the structure of the open pore remains elusive. Furthermore, although many residues have been implicated in altering the selectivity of CFTR, the structure of the \"selectivity filter\" has yet to be determined. In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. This selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer.

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a \"ball-and-chain\" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

The transcription factor farnesoid X receptor (FXR) regulates energy metabolism. Specifically, FXR functions to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion in intestinal epithelial cells. Therefore, this study aimed to investigate the role of FXR in CFTR-mediated Cl- secretion in renal tubular cells and to further elucidate its effects on renal cyst formation and growth. CFTR-mediated Cl- transport was evaluated via short-circuit current (ISC) measurements in Madin-Darby canine kidney (MDCK) cell monolayers and primary rat inner medullary collecting duct cells. The role of FXR in renal cyst formation and growth was determined by the MDCK cell-derived cyst model. Incubation with synthesized (GW4064) and endogenous (CDCA) FXR ligands reduced CFTR-mediated Cl- secretion in a concentration- and time-dependent manner. The inhibitory effect of FXR ligands was not due to the result of reduced cell viability and was attenuated by cotreatment with an FXR antagonist. FXR activation significantly decreased CFTR protein but not its mRNA. In addition, FXR activation inhibited CFTR-mediated Cl- secretion in primary renal collecting duct cells. FXR activation decreased ouabain-sensitive ISC without altering Na+-K+-ATPase mRNA and protein levels. Furthermore, FXR activation significantly reduced the number of cysts and renal cyst expansion. These inhibitory effects were correlated with a decrease in the expression of protein synthesis regulators mammalian target of rapamycin/S6 kinase. This study shows that FXR activation inhibits Cl- secretion in renal cells via inhibition of CFTR expression and retards renal cyst formation and growth. The discoveries point to a physiological role of FXR in the regulation of CFTR and a potential therapeutic application in polycystic kidney disease treatment.NEW & NOTEWORTHY The present study reveals that farnesoid X receptor (FXR) activation reduces microcyst formation and enlargement. This inhibitory effect of FXR activation is involved with decreased cell proliferation and cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion in renal collecting duct cells. FXR might represent a novel target for the treatment of autosomal dominant polycystic kidney disease.

OBJECTIVE: Myotonia congenita (MC) is a muscle channelopathy in which pathogenic variants in a key sarcolemmal chloride channel Gene (CLCN1) cause myotonia. This study used muscle magnetic resonance imaging (MRI) to quantify contractile properties and fat replacement of muscles in a Danish cohort of MC patients.
METHODS: Individuals with the Thomsen (dominant) and Becker (recessive) variants of MC were studied. Isometric muscle strength, whole-body MRI, and clinical data were collected. The degree of muscle fat replacement of thigh, calf, and forearm muscles was quantitively calculated on Dixon MRI as fat fractions (FFs). Contractility was evaluated as the muscle strength per contractile muscle cross-sectional area (PT/CCSA). Muscle contractility was compared with clinical data.
RESULTS: Intramuscular FF was increased and contractility reduced in calf and in forearm muscles compared with controls (FF = 7.0-14.3% vs. 5.3-9.6%, PT/CCSA = 1.1-4.9 Nm/cm2 vs. 1.9-5.8 Nm/cm2 [p < 0.05]). Becker individuals also showed increased intramuscular FF and reduced contractility of thigh muscles (FF = 11.9% vs. 9.2%, PT/CCSA = 1.9 Nm/cm2 vs. 3.2 Nm/cm2 [p < 0.05]). Individual muscle analysis showed that increased FF was limited to seven of 18 examined muscles (p < 0.05). There was a weak correlation between reduced contractility and severity of symptoms.
CONCLUSIONS: Individuals with MC have increased fat replacement and reduced contractile properties of muscles. Nonetheless, changes were small and likely did not impact clinically on their myotonic symptoms.

Chloride (Cl-) is considered a crucial nutrient for plant growth, but it can be a challenge under saline conditions. Excessive accumulation of Cl- in leaves can cause toxicity. Chloride channels (CLCs) are expressed in the inner membranes of plant cells and function as essential Cl- exchangers or channels. In response to salt stress in plants, CLCs play a crucial role, and CLC proteins assist in maintaining the intracellular Cl- homeostasis by sequestering Cl- into vacuoles. Sodium chloride (NaCl) is the primary substance responsible for causing salt-induced phytotoxicity. However, research on plant responses to Cl- stress is comparatively rare, in contrast to that emphasizing Na+. This review provides a comprehensive overview of the plant response and tolerance to Cl- stress, specifically focusing on comparative analysis of CLC protein structures in different species. Additionally, to further gain insights into the underlying mechanisms, the study summarizes the identified CLC genes that respond to salt stress. This review provides a comprehensive overview of the response of CLCs in terrestrial plants to salt stress and their biological functions, aiming to gain further insights into the mechanisms underlying the response of CLCs in plants to salt stress.

Cl- influx and efflux through Cl- channels play a role in regulating the homeostasis of biological functions. Therefore, the hyperfunction or dysfunction of Cl- channels elicits pathological mechanisms. The Cl- channel superfamily includes voltage-gated Cl- (ClC) channels, Ca2+-activated Cl- channels (ClCa; TMEM16A/TMEM16B), cystic fibrosis transmembrane conductance regulator channels, and ligand-gated Cl- channels. These channels are ubiquitously expressed to regulate ion homeostasis, muscle tonus, membrane excitability, cell volume, survival, neurotransmission, and transepithelial transport. The activation or inhibition of Cl- channels changes the membrane potential, thereby affecting cytosolic Ca2+ signals. An elevation in cytosolic [Ca2+] triggers physiological and pathological responses in most cells. However, the roles of Cl- channels have not yet been examined as extensively as cation (Na+, Ca2+, and K+) channels. We recently reported the functional expression of: (i) TMEM16A/ClCa channels in portal vein and pulmonary arterial smooth muscle cells (PASMC), pinealocytes, and brain capillary endothelial cells; (ii) TMEM16B/ClCa channels in pinealocytes; (iii) ClC-3 channels in PASMC and chondrocytes; and (iv) ClC-7 channels in chondrocytes. We also showed that the down-regulation of TMEM16A and ClC-7 channel expression was associated with cirrhotic portal hypertension and osteoarthritis, respectively, whereas the enhanced expression of TMEM16A and ClC-3 channels was involved in the pathogenesis of cerebral ischemia and pulmonary arterial hypertension, respectively. Further investigations on the physiological/pathological functions of Cl- channels will provide insights into biological functions and contribute to the screening of novel target(s) of drug discovery for associated diseases.

In a recent study, Guo and colleagues characterised the function of an elusive endoplasmic reticulum (ER) anion channel protein, Chloride Channel CLiC Like 1 (CLCC1), and identified rare CLCC1 variants in people with amyotrophic lateral sclerosis (ALS). CLCC1 mutants disrupted ER function in vitro and promoted ALS-like pathology and neurodegeneration in mice. This work reveals a previously uncharacterised pathway involved in ER calcium release and highlights new pathogenic mechanisms underlying neurodegeneration.