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.

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.

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

Myotonia congenita is a hereditary muscle disease mainly characterized by muscle hyperexcitability, which leads to a sustained burst of discharges that correlates with the magnitude and duration of involuntary aftercontractions, muscle stiffness, and hypertrophy. Mutations in the chloride voltage-gated channel 1 (CLCN1) gene that encodes the skeletal muscle chloride channel (ClC-1) are responsible for this disease, which is commonly known as myotonic chloride channelopathy. The biophysical properties of the mutated channel have been explored and analyzed through in vitro approaches, providing important clues to the general function/dysfunction of the wild-type and mutated channels. After an exhaustive search for CLCN1 mutations, we report in this review more than 350 different mutations identified in the literature. We start discussing the physiological role of the ClC-1 channel in skeletal muscle functioning. Then, using the reported functional effects of the naturally occurring mutations, we describe the biophysical and structural characteristics of the ClC-1 channel to update the knowledge of the function of each of the ClC-1 helices, and finally, we attempt to point out some patterns regarding the effects of mutations in the different helices and loops of the protein.

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.

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. 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 mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and 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 apo conformations of CLC-2 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 from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any 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 show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data 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.

Volume-regulated anion channels (VRACs) are hexamers of LRRC8 proteins that are crucial for cell volume regulation. N termini (NTs) of the obligatory LRRC8A subunit modulate VRACs activation and ion selectivity, but the underlying mechanisms remain poorly understood. Here, we report a 2.8-Å cryo-electron microscopy structure of human LRRC8A that displays well-resolved NTs. Amino-terminal halves of NTs fold back into the pore and constrict the permeation path, thereby determining ion selectivity together with an extracellular selectivity filter with which it works in series. They also interact with pore-surrounding helices and support their compact arrangement. The C-terminal halves of NTs interact with intracellular loops that are crucial for channel activation. Molecular dynamics simulations indicate that low ionic strength increases NT mobility and expands the radial distance between pore-surrounding helices. Our work suggests an unusual pore architecture with two selectivity filters in series and a mechanism for VRAC activation by cell swelling.

Hereditary myotonia (HM) is characterized by delayed muscle relaxation after contraction as a result of a mutation in the CLCN1 gene. We describe here a complex CLCN1 variant in a mixed-breed dog with clinical and electromyographic signs of HM. Blood samples from the myotonic dog, as well as from his male littermate and parents, were analyzed via amplification of the 23 exons encoding CLCN1. After sequencing the CLCN1 gene, a complex variant was found in exon 6 c.[705T>G; 708del; 712_732del], resulting in a premature stop codon in exon 7 and a protein that was 717 amino acids shorter than the normal CLC protein. The myotonic dog was identified as homozygous recessive for the complex CLCN1 variant; its parents were heterozygous, and its male littermate was homozygous wild-type. Knowledge of the CLCN1 mutations responsible for the development of hereditary myotonia allows greater clarification of this condition.