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Molecules (Basel, Switzerland) Nov 2020Synthetic anion transporters that facilitate chloride transport are promising candidates for channelopathy treatments. However, most anion transporters exhibit an...
Synthetic anion transporters that facilitate chloride transport are promising candidates for channelopathy treatments. However, most anion transporters exhibit an undesired side effect of facilitating proton transport via interacting with fatty acids present in the membrane. To address the limitation, we here report the use of a new tetrapodal scaffold to maximize the selective interaction with spherical chloride over binding the carboxylate headgroup of fatty acids. One of the new transporters demonstrated a high selectivity for chloride uniport over fatty acid-induced proton transport while being >10 times more active in chloride uniport than strapped calixpyrroles that were previously the only class of compounds known to possess similar selectivity properties.
Topics: Anion Transport Proteins; Anions; Chlorides; Chromatography, Thin Layer; Crystallography, X-Ray; Fatty Acids; Ion Transport; Lipid Bilayers; Magnetic Resonance Spectroscopy; Nitrates; Pyrenes; Sulfonic Acids
PubMed: 33172141
DOI: 10.3390/molecules25215179 -
Pharmacological Research Dec 2023The NLRP3 inflammasome is a supramolecular complex that is linked to sterile and pathogen-dependent inflammation, and its excessive activation underlies many diseases....
The NLRP3 inflammasome is a supramolecular complex that is linked to sterile and pathogen-dependent inflammation, and its excessive activation underlies many diseases. Ion flux disturbance and cell volume regulation are both reported to mediate NLRP3 inflammasome activation, but the underlying orchestrating signaling remains not fully elucidated. The volume-regulated anion channel (VRAC), formed by LRRC8 proteins, is an important constituent that controls cell volume by permeating chloride and organic osmolytes in response to cell swelling. We now demonstrate that Lrrc8a, the essential component of VRAC, plays a central and specific role in canonical NLRP3 inflammasome activation. Moreover, VRAC acts downstream of K efflux for NLRP3 stimuli that require K efflux. Mechanically, our data demonstrate that VRAC modulates itaconate efflux and damaged mitochondria production for NLRP3 inflammasome activation. Further in vivo experiments show mice with Lrrc8a deficiency in myeloid cells were protected from lipopolysaccharides (LPS)-induced endotoxic shock. Taken together, this work identifies VRAC as a key regulator of NLRP3 inflammasome and innate immunity by regulating mitochondrial adaption for macrophage activation and highlights VRAC as a prospective drug target for the treatment of NLRP3 inflammasome and itaconate related diseases.
Topics: Mice; Animals; Membrane Proteins; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Anions; Mitochondria
PubMed: 38006980
DOI: 10.1016/j.phrs.2023.107016 -
Journal of Intensive Care Medicine Dec 2022Previous studies evaluating patients in the Intensive Care Unit with lactic acidosis determined that the anion gap is an insensitive screening tool for elevated blood...
Previous studies evaluating patients in the Intensive Care Unit with lactic acidosis determined that the anion gap is an insensitive screening tool for elevated blood lactate. No prior study has examined the relationship between anion gap and serum lactate within the first hours of the development of lactic acidosis. Data were obtained prospectively from a convenience sample of adult trauma patients at a single level 1 trauma center. Venous samples were drawn prior to initiation of intravenous fluid resuscitation. A linear regression model was constructed to assess the relationship between serum lactate and anion gap, and 95% prediction intervals were computed. Logistic regression models were constructed to determine the sensitivity and specificity for several different anion gap and lactate cutpoints. 128 patients with elevated serum lactate levels (>2.1 mmol/L) and 63 patients with normal serum lactate levels ( 2.1 mmol/L) were included. The sensitivity of an elevated anion gap (> 10) to reveal hyperlactatemia was only 43% whereas specificity was 84%. Sensitivity improved if the upper limit of normal anion gap was lowered and with increasing levels of serum lactate. The coefficient of determination between serum lactate level and AG yielded an R of 0.30 (p < 0.001) and the slope of this relationship was 2.185 with a 95% confidence interval of 2.011-2.359. The mean 95% prediction interval was 8.9. Within the first hour of the development of lactic acidosis due to hypovolemic shock, the anion gap was not a sensitive indicator of an elevated serum lactate level, but it was fairly specific. The anion gap increased to a greater extent than the serum lactate, the 95% mean prediction interval was wide and approximately 70% of the change in anion gap could not be explained by increases in serum lactate, suggesting that other anions contribute to the anion gap in lactic acidosis.
Topics: Adult; Humans; Acidosis, Lactic; Acid-Base Equilibrium; Lactic Acid; Shock; Anions
PubMed: 35668631
DOI: 10.1177/08850666221106413 -
Nature Communications Aug 2023Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels...
Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. Arabidopsis thaliana CLCa (AtCLCa) transporter localizes to the tonoplast which imports NO and to a less extent Cl from cytoplasm. The activity of AtCLCa and many other CLCs is regulated by nucleotides and phospholipids, however, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with NO and Cl, respectively. Both structures are captured in ATP and PI(4,5)P bound conformation. Structural and electrophysiological analyses reveal a previously unidentified N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ- phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance. Our data further show that PI(4,5)P or PI(3,5)P binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios and provides new insights for future study of CLCs.
Topics: Arabidopsis; Nucleotides; Protons; Nitrates; Phospholipids; Arabidopsis Proteins; Anions; Adenosine Triphosphate; Chloride Channels
PubMed: 37573431
DOI: 10.1038/s41467-023-40624-z -
Molecules (Basel, Switzerland) Jun 2022Cyclic pentaphenylalanine was studied as an efficient anion sensor for halides, thiocyanate and oxoanions in acetonitrile and methanol. Stability constants of the...
Cyclic pentaphenylalanine was studied as an efficient anion sensor for halides, thiocyanate and oxoanions in acetonitrile and methanol. Stability constants of the corresponding complexes were determined by means of fluorimetric, spectrophotometric, H NMR, and microcalorimetric titrations. A detailed structural overview of receptor-anion complexes was obtained by classical molecular dynamics (MD) simulations. The results of H NMR and MD studies indicated that the bound anions were coordinated by the amide groups of cyclopeptide, as expected. Circular dichroism (CD) titrations were also carried out in acetonitrile. To the best of our knowledge, this is the first example of the detection of anion binding by cyclopeptide using CD spectroscopy. The CD spectra were calculated from the structures obtained by MD simulations and were qualitatively in agreement with the experimental data. The stoichiometry of almost all complexes was 1:1 (receptor:anion), except for dihydrogen phosphate where the binding of dihydrogen phosphate dimer was observed in acetonitrile. The affinity of the cyclopeptide receptor was correlated with the structure of anion coordination sphere, as well as with the solvation properties of the examined solvents.
Topics: Acetonitriles; Anions; Molecular Dynamics Simulation; Peptides, Cyclic; Phosphates
PubMed: 35745042
DOI: 10.3390/molecules27123918 -
Nature Mar 2022The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes. To remedy this, charged...
The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes. To remedy this, charged amphiphilic molecules have been classically used as carriers. However, such amphiphilic carriers may cause aggregation and non-specific membrane lysis. Here we show that globular dodecaborate clusters, and prominently BBr, can function as anionic inorganic membrane carriers for a broad range of hydrophilic cargo molecules (with molecular mass of 146-4,500 Da). We show that cationic and neutral peptides, amino acids, neurotransmitters, vitamins, antibiotics and drugs can be carried across liposomal membranes. Mechanistic transport studies reveal that the carrier activity is related to the superchaotropic nature of these cluster anions. We demonstrate that BBr affects cytosolic uptake of different small bioactive molecules, including the antineoplastic monomethyl auristatin F, the proteolysis targeting chimera dBET1 and the phalloidin toxin, which has been successfully delivered in living cells for cytoskeleton labelling. We anticipate the broad and distinct delivery spectrum of our superchaotropic carriers to be the starting point of conceptually distinct cell-biological, neurobiological, physiological and pharmaceutical studies.
Topics: Anions; Biological Transport; Boron; Cations; Drug Carriers; Hydrophobic and Hydrophilic Interactions; Peptides; Pharmaceutical Preparations
PubMed: 35322251
DOI: 10.1038/s41586-022-04413-w -
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 -
Neurochemical Research Jan 2022Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly... (Review)
Review
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by excitatory amino acid transporters (EAATs) 1-5, a subfamily of glutamate transporters. The five proteins utilize a complex transport stoichiometry that couples glutamate transport to the symport of three Na ions and one H in exchange with one K to accumulate glutamate against up to 10-fold concentration gradients. They are also anion-selective channels that open and close during transitions along the glutamate transport cycle. EAATs belong to a larger family of secondary-active transporters, the SLC1 family, which also includes purely Na- or H-coupled prokaryotic transporters and Na-dependent neutral amino acid exchangers. In recent years, molecular cloning, heterologous expression, cellular electrophysiology, fluorescence spectroscopy, structural approaches, and molecular simulations have uncovered the molecular mechanisms of coupled transport, substrate selectivity, and anion conduction in EAAT glutamate transporters. Here we review recent findings on EAAT transport mechanisms, with special emphasis on the highly conserved hairpin 2 gate, which has emerged as the central processing unit in many of these functions.
Topics: Amino Acid Transport System X-AG; Animals; Anions; Biological Transport; Excitatory Amino Acid Transporter 1; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid; Mammals
PubMed: 33587237
DOI: 10.1007/s11064-021-03252-x -
Journal of the American Chemical Society Jun 2022Ion transport across lipid membranes in biology is controlled by stimuli-responsive membrane channels and molecular machine ion pumps such as ATPases. Here, we report a...
Ion transport across lipid membranes in biology is controlled by stimuli-responsive membrane channels and molecular machine ion pumps such as ATPases. Here, we report a synthetic molecular machine-like ion transport relay, in which transporters on opposite sides of a lipid bilayer membrane facilitate transport by passing ions between them. By incorporating a photo-responsive telescopic arm into the relay design, this process is reversibly controlled in response to irradiation with blue and green light. Transport occurs only in the extended state when the length of the arm is sufficient to pass the anion between transporters located on opposite sides of the membrane. In contrast, the contracted state of the telescopic arm is too short to mediate effective transport. The system acts as a stimuli-responsive ensemble of machine-like components, reminiscent of robotic arms in a factory assembly line, working cooperatively to mediate ion transport. This work points to new prospects for using lipid bilayer membranes as scaffolds for confining, orientating, and controlling the relative positions of molecular machines, thus enabling multiple components to work in concert and opening up new applications in biological contexts.
Topics: Anions; Biological Transport; Ion Channels; Ion Transport; Lipid Bilayers
PubMed: 35652660
DOI: 10.1021/jacs.2c02612 -
Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly.ELife Mar 2023Volume-regulated anion channels (VRACs) mediate volume regulatory Cl and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E...
Volume-regulated anion channels (VRACs) mediate volume regulatory Cl and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL1) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL1) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL1) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.
Topics: Membrane Proteins; Cryoelectron Microscopy; Biological Transport; Anions
PubMed: 36897307
DOI: 10.7554/eLife.82431