Study on Concrete Deterioration in Different NaCl-Na2SO4 Solutions and the Mechanism of Cl− Diffusion

The diffusion of sulfate (SO₄²⁻) and chloride (Cl⁻) ions from rivers, salt lakes and saline soil into reinforced concrete is one of the main factors that contributes to the corrosion of steel reinforcing bars, thus reducing their mechanical properties. This work experimentally investigated the corrosion process involving various concentrations of NaCl-Na₂SO₄ leading to the coupled erosion of concrete. The appearance, weight, and mechanical properties of the concrete were measured throughout the erosion process, and the Cl⁻ and SO₄²⁻ contents in concrete were determined using Cl⁻ rapid testing and spectrophotometry, respectively. Scanning electron microscopy, energy spectrometry, X-ray diffractometry, and mercury porosimetry were also employed to analyze microstructural changes and complex mineral combinations in these samples. The results showed that with higher Na₂SO₄ concentration and longer exposure time, the mass, compressive strength, and relative dynamic elastic modulus gradually increased and large pores gradually transitioned to medium and small pores. When the Na₂SO₄ mass fraction in the salt solution was ≥10 wt%, there was a downward trend in the mechanical properties after exposure for a certain period of time. The Cl⁻ diffusion rate was thus related to Na₂SO₄ concentration. When the Na₂SO₄ mass fraction in solution was ≤5 wt% and exposure time short, SO₄²⁻ and cement hydration/corrosion products hindered Cl⁻ migration. In a concentrated Na₂SO₄ environment (≥10 wt%), the Cl⁻ diffusion rate was accelerated in the later stages of exposure. These experiments further revealed that the Cl⁻ migration rate was higher than that of SO₄²⁻.     

Neto2 is a KCC2 interacting protein required for neuronal Cl? regulation in hippocampal neurons

KCC2 is a neuron-specific K⁺–Cl⁻ cotransporter that is essential for Cl⁻ homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl⁻ extrusion, resulting in decreased synaptic inhibition in hippocampal neurons.     

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na⁺ absorption, a defective cAMP-dependent Cl⁻ conductance, higher concentrations of Na⁺, Cl⁻, K⁺, and Ca²⁺ in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca²⁺ entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.     

CLC Cl /H+ transporters constrained by covalent cross-linking

CLC Cl⁻/H⁺ exchangers are homodimers with Cl⁻-binding and H⁺-coupling residues contained within each subunit. It is not known whether the transport mechanism requires conformational rearrangement between subunits or whether each subunit operates as a separate exchanger. We designed various cysteine substitution mutants on a cysteine-less background of CLC-ec1, a bacterial CLC exchanger of known structure, with the aim of covalently linking the subunits. The constructs were cross-linked in air or with exogenous oxidant, and the cross-linked proteins were reconstituted to assess their function. In addition to conventional disulfides, a cysteine–lysine cross-bridge was formed with I₂ as an oxidant. The constructs, all of which contained one, two, or four cross-bridges, were functionally active and kinetically competent with respect to Cl⁻ turnover rate, Cl⁻/H⁺ exchange stoichiometry, and H⁺ pumping driven by a Cl⁻ gradient. These results imply that large quaternary rearrangements, such as those known to occur for “common gating” in CLC channels, are not necessary for the ion transport cycle and that it is therefore likely that the transport mechanism is carried out by the subunits working individually, as with “fast gating” of the CLC channels.     

Etiology of distinct membrane excitability in pre- and posthearing auditory neurons relies on activity of Cl? channel TMEM16A

The developmental rehearsal for the debut of hearing is marked by massive changes in the membrane properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Whereas the underlying mechanisms for the developing HC transition to mature stage are understood in detail, the maturation of SGNs from hyperexcitable prehearing to quiescent posthearing neurons with broad dynamic range is unknown. Here, we demonstrated using pharmacological approaches, caged-Ca²⁺ photolysis, and gramicidin patch recordings that the prehearing SGN uses Ca²⁺-activated Cl⁻ conductance to depolarize the resting membrane potential and to prime the neurons in a hyperexcitable state. Immunostaining of the cochlea preparation revealed the identity and expression of the Ca²⁺-activated Cl⁻ channel transmembrane member 16A (TMEM16A) in SGNs. Moreover, null deletion of TMEM16A reduced the Ca²⁺-activated Cl⁻ currents and action potential firing in SGNs. To determine whether Cl⁻ ions and TMEM16A are involved in the transition between pre- and posthearing features of SGNs we measured the intracellular Cl⁻ concentration [Cl⁻]_i in SGNs. Surprisingly, [Cl⁻]_i in SGNs from prehearing mice was ∼90 mM, which was significantly higher than posthearing neurons, ∼20 mM, demonstrating discernible altered roles of Cl⁻ channels in the developing neuron. The switch in [Cl⁻]_i stems from delayed expression of the development of intracellular Cl⁻ regulating mechanisms. Because the Cl⁻ channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of SGN action potentials, developmental alteration of [Cl⁻]_i, and hence the equilibrium potential for Cl⁻ (E_Cl), transforms pre- to posthearing phenotype.The dynamic range of neuronal action potentials (APs) resides within voltages that are outside the reversal potentials (E_rev) of most ion currents except Cl⁻ currents, making Cl⁻ conductance the most versatile one in a course of a single AP. Neurons use this adaptable feature of Cl⁻ conductance with respect to the resting membrane potential (RMP) of neurons to confer synaptic plasticity by altering intracellular Cl⁻ (Cl⁻_i) homeostasis during development. This process transforms depolarizing GABA/glycinergic-mediated responses in immature to hyperpolarizing responses in mature neurons (1, 2). A similar synaptic switch has been described in auditory brainstem neurons, where the mature GABA/glycinergic-induced inhibitory neurotransmission contributes strongly toward the computation of interaural level and time differences required for sound source localization (3–6). The depolarization mediated by GABA/glycine in early postnatal development may increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), which is predicted to promote synapse stabilization in the CNS (1). We hypothesized that besides synaptic plasticity one mechanism that alters the firing phenotype of developing neurons is via changes in intracellular Cl⁻ concentration ([Cl]_i) and activation of voltage and Ca²⁺-activated Cl⁻ channels (CaCCs).CaCCs are encoded by anoctamin 1 and 2 (ANO1 and 2), also known as transmembrane member 16A and B (TMEM16A and B) genes, which are expressed in epithelia and smooth muscle cells (7, 8) and in sensory cells such as nociceptive dorsal root ganglion neurons (9, 10), cilia of olfactory cells (11), and in rods and cones (12). The prevailing functions of CaCCs are ascribed to the amplification of pain sensation (10), cone responses (12), and olfactory signal transduction (13, 14), although recent reports using TMEM16B knockout mice suggest that CaCCs may play a limited role in signal amplification of olfactory transduction (11). TMEM16A has been identified in the cochlea in a cell-type-specific manner, showing robust labeling in basal cells of the stria vascularis and efferent endings of the auditory nerve (15), but its role in the inner ear has not been determined.The trademark of the developing auditory neuron is the rhythmic and burst-patterned spontaneous AP (SAP), which is thought to shape synapse formation and refinement in the brainstem (16, 17). In the inner ear, inputs from Ca²⁺-mediated SAPs from developing hair cells (HCs) sculpt the firing patterns of spiral ganglion neurons (SGNs) (18, 19). However, SGNs evolve from depolarizing hyperexcitable to hyperpolarized mature neurons with a wide dynamic range (20). Mechanisms underlying the remarkable changes in SGN phenotype during development are not well understood. Here, we demonstrate the origin and molecular mechanisms of the transition from primordial to mature auditory neurons. SGNs undergo marked alterations in intracellular Cl⁻ concentration ([Cl⁻]_i) handling during development and in doing so transform a predominantly inwardly driven Cl⁻ current into outwardly directed current through activation of TMEM16 channels.     

Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: An unexpected role for intracellular chloride channels

GEF1 is a gene in Saccharomyces cerevisiae, which encodes a putative voltage-regulated chloride channel. gef1 mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p. The defect is due to an inability to transfer Cu⁺ to apoFet3p within the secretory apparatus. We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl⁻. Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl⁻ there is very little Cu-loading at any pH. Cl⁻ has a positive allosteric effect on Cu-loading of apoFet3p. Kinetic studies suggest that Cl⁻ may also bind to Fet3p and that Cu⁺ has an allosteric effect on the binding of Cl⁻ to the enzyme. Thus, Cl⁻ may be required for the metal loading of proteins within the secretory apparatus. These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.     

The Cap1–claudin-4 regulatory pathway is important for renal chloride reabsorption and blood pressure regulation

The paracellular pathway through the tight junction provides an important route for transepithelial chloride reabsorption in the kidney, which regulates extracellular salt content and blood pressure. Defects in paracellular chloride reabsorption may in theory cause deregulation of blood pressure. However, there is no evidence to prove this theory or to demonstrate the in vivo role of the paracellular pathway in renal chloride handling. Here, using a tissue-specific KO approach, we have revealed a chloride transport pathway in the kidney that requires the tight junction molecule claudin-4. The collecting duct-specific claudin-4 KO animals developed hypotension, hypochloremia, and metabolic alkalosis due to profound renal wasting of chloride. The claudin-4–mediated chloride conductance can be regulated endogenously by a protease—channel-activating protease 1 (cap1). Mechanistically, cap1 regulates claudin-4 intercellular interaction and membrane stability. A putative cap1 cleavage site has been identified in the second extracellular loop of claudin-4, mutation of which abolished its regulation by cap1. The cap1 effects on paracellular chloride permeation can be extended to other proteases such as trypsin, suggesting a general mechanism may also exist for proteases to regulate the tight junction permeabilities. Together, we have discovered a theory that paracellular chloride permeability is physiologically regulated and essential to renal salt homeostasis and blood pressure control.Chloride is the most abundant extracellular anion and thereby determines extracellular fluid volume (ECFV) and blood pressure (BP) (1, 2). The kidney plays a vital role in ECFV and BP control through complex regulatory mechanisms acting upon ion channels and transporters located in the aldosterone-sensitive distal nephron (ASDN) (3, 4). The ASDN comprises the distal convoluted tubule (DCT), the connecting tubule (CNT), and the collecting duct (CD). The primary chloride transport mechanism in the DCT is through the thiazide-sensitive Na⁺/Cl⁻ cotransporter (NCC) to reabsorb Cl⁻ coupled with equal moles of Na⁺ (5). The chloride transport mechanism in the CNT and CD, on the other hand, has been under debate for many years. Recent advances have identified an electroneutral transport pathway for chloride using the Cl⁻/bicarbonate exchanger (Slc26a4; pendrin) (6) and the Na⁺-driven Cl⁻/bicarbonate exchanger (Slc4a8; NDCBE) (7) in the β-intercalated cells (ICs) of CNT and CD. However, such an electroneutral pathway is not able to couple Cl⁻ reabsorption with epithelial sodium channel (ENaC)-based Na⁺ reabsorption that takes place in the principal cells (PCs) of CNT and CD (8), despite many efforts to connect ENaC and pendrin gene expression through endocrine or paracrine mechanisms (9, 10). We and others have previously demonstrated the presence of a paracellular Cl⁻ pathway or “chloride shunt” in vitro in the CNT and CD cells (11, 12). The paracellular Cl⁻ channel is made of a key claudin molecule, claudin-4, within the tight junction (TJ) (11). Here, using a tissue-specific KO approach, we have provided conclusive evidence that claudin-4 is required for renal reabsorption of Cl⁻; loss of claudin-4 in a transgenic mouse model claudin-4^flox/flox/Aqp2^Cre caused significant renal wasting of chloride, sodium, and volume.The claudins are a 28-member family of tetraspan proteins that range in molecular mass from 20 to 28 kDa and form a class of ion channels oriented perpendicular to the membrane plane and connecting two extracellular compartments, known as the paracellular channel (13). Claudins associate by cis interactions within the plasma membrane of the same cell followed by trans interactions between neighboring cells to assemble them in the TJ. Previously, we have observed the cis interaction between claudin-4 and claudin-8 and demonstrated that their interaction was required for TJ assembly (11). How claudins trans interact, on the other hand, is poorly understood. Here, using several biochemical and imaging criteria, we have found a protease, channel-activating protease 1 (cap1), that transiently disrupted the claudin-4 interaction. Loss of claudin-4 interaction reduced its plasma membrane stability and abundance. Cap1 was the first of several membrane-tethered serine proteases found to activate the amiloride-sensitive ENaC in the ASDN (14, 15). In aldosterone-infused animals, the renal expression levels of cap1 were profoundly elevated, accompanied by increases in the CD Na⁺ uptake (16). Despite considerable evidence regarding cap1 regulation of the Na⁺ transport in ASDN, it is not known what role cap1 may play in the Cl⁻ handling. Here, using molecular and electrophysiological approaches, we have revealed how cap1 regulates claudin-4–dependent paracellular Cl⁻ permeation in the CD.     

Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach

Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9^−/−) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. Immunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9^−/− mice, in contrast to the predominant cytoplasmic localization in Slc26a9^+/+ mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9^−/− stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl⁻/HCO₃⁻ exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/secretory canaliculi and by regulating chloride secretion in parietal cells.     

Structure and charging of hydrophobic material/water interfaces studied by phase-sensitive sum-frequency vibrational spectroscopy

We have studied the hydrophobic water/octadecyltrichlorosilane (OTS) interface by using the phase-sensitive sum-frequency vibrational spectroscopy (PS-SFVS), and we obtained detailed structural information of the interface at the molecular level. Excess ions emerging at the interface were detected by changes of the surface vibrational spectrum induced by the surface field created by the excess ions. Both hydronium (H₃O⁺) and hydroxide (OH⁻) ions were found to adsorb at the interface, and so did other negative ions such as Cl⁻. By varying the ion concentrations in the bulk water, their adsorption isotherms were measured. It was seen that among the three, OH⁻ has the highest adsorption energy, and H₃O⁺ has the lowest; OH⁻ also has the highest saturation coverage, and Cl⁻ has the lowest. The result shows that even the neat water/OTS interface is not neutral, but charged with OH⁻ ions. The result also explains the surprising observation that the isoelectric point appeared at ∼3.0 when HCl was used to decrease the pH starting from neat water.     

A single serine residue controls the cation dependence of substrate transport by the rat serotonin transporter

The serotonin transporter (SERT) is a member of the Na⁺/Cl⁻-dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. Here, replacement of serine-545 in the recombinant rat SERT by alanine was found to alter the cation dependence of serotonin uptake. Substrate transport was now driven as efficiently by LiCl as by NaCl without significant changes in serotonin affinity. Binding of the antidepressant [³H]imipramine occurred with 1/5th the affinity, whereas [³H]citalopram binding was unchanged. These results indicate that serine-545 is a crucial determinant of both the cation dependence of serotonin transport by SERT and the imipramine binding properties of SERT.     

Intracellular Cl? as a signaling ion that potently regulates Na+/HCO3? transporters

Cl⁻ is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl⁻ (Cl⁻_in) as a signaling ion has not been previously evaluated. Here we report that Cl⁻_in functions as a regulator of cellular Na⁺ and HCO₃⁻ concentrations and transepithelial transport through modulating the activity of several electrogenic Na⁺-HCO₃⁻ transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl⁻_in concentrations highlighting the role of GXXXP motifs in Cl⁻ sensing. Regulation of the ubiquitous Na⁺-HCO3⁻ co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl⁻_in interacting at a low affinity GXXXP-containing site. IP₃ receptor binding protein released with IP₃ (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl⁻_in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl⁻_in interacting site. NBCe1-A is unaffected by Cl⁻_in between 5 and 140 mM. However, deletion of NBCe1-A residues 29–41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl⁻_in. These findings reveal a cellular Cl⁻_in sensing mechanism that plays an important role in the regulation of Na⁺ and HCO₃⁻ transport, with critical implications for the role of Cl⁻ in cellular ion homeostasis and epithelial fluid and electrolyte secretion.Cl⁻ and HCO₃⁻ are the two major intracellular anions in mammalian cells. Specific transporters, channels, and the membrane potential tightly regulate their extracellular and intracellular concentrations. In turn, Cl⁻ and HCO₃⁻ regulate the concentration of other ions, including Na⁺, K⁺, and SO₄²⁻, either directly or indirectly. Known ubiquitous Cl⁻- and HCO₃⁻-coupled transporters include the NaCl cotransporters NCCs, the KCl cotransporters KCCs, the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 (1), the SLC26 Cl⁻/HCO₃⁻ exchangers and channels (2, 3), and members of the SLC4 exchangers and cotransporters family (4). The intracellular Cl⁻ (Cl⁻_in) concentration is also regulated by the ClCs (5) and Anoctamines Cl⁻ channels (6). Cl⁻ plays a role in a wide variety of cellular transport functions, including regulation of the membrane potential (6), cell volume (2), systemic and cellular acid–base balance (4), and transepithelial fluid and electrolyte secretion (7). In addition, Cl⁻ was reported to regulate transient receptor potential (TRP) channels (8), receptors assembly and function (9–11), activation of Neutrophil β2 Integrins (12), and the cell cycle (13).Like Cl⁻, HCO₃⁻ also has many important physiological roles, being the principal biological pH buffer (7) and an activator of the soluble adenylyl cyclase (14). In epithelia, HCO₃⁻ has a key role in tissue/cell viability. Among other fundamental roles, HCO₃⁻ drives Cl⁻ absorption and fluid secretion, stimulates mucin secretion, and controls solubilization of secreted macromolecules (7). Epithelial HCO₃⁻ secretion is fueled by the cellular Na⁺ gradient, which provides the driving force for HCO₃⁻ entry across the basolateral membrane mediated by the Na⁺-HCO₃⁻ cotransport NBCe1-B. HCO₃⁻ then exits the luminal membrane by the coordinated and coupled functions of the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR) and the electrogenic Cl⁻/HCO₃⁻ exchanger slc26a6 (3, 7, 15). In the kidney, NBCe1-A mediates basolateral HCO₃⁻ extrusion (16, 17). In secretory ducts the basolateral NBCe1-B–mediated HCO₃⁻ influx is coupled to apical HCO₃⁻ secretion and Cl⁻ absorption via CFTR and slc26a6 (7). Cl⁻_in is reduced along the ducts as luminal Cl⁻ is reduced and luminal HCO₃⁻ is increased (18, 19).Members of the SLC4 superfamily of HCO₃⁻ cotransporters are key transporters involved in cellular HCO₃⁻ and Cl⁻ homeostasis (4, 7). The family consists of several subfamilies, including the electrogenic NBCe1 and NBCe2, electroneutral NBCn1 and NBCn2, Cl⁻-coupled anion exchangers AEs, and the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger NDCBE (4). In a wide variety of tissues, NBCe1-B mediates the electrogenic transport of 1Na⁺ and 2HCO₃⁻ ions (likely Na⁺-CO₃²⁻) (4, 17) and functions as the main epithelial HCO₃⁻ entry mechanism in the basolateral membrane of polarized cells (7). Cell-specific electrogenic NBC transporters include NBCe1-A, which is expressed in the basolateral membrane of the renal proximal tubule (4, 17), and NBCe2-C, which is found in the choroid plexus (20). IRBIT, which regulates NBCe1-B (21–23) and NBCn1-A (24), binds to the IP₃ binding domain of the IP₃ receptors (IP₃Rs) (25). IRBIT is released from the IP₃Rs upon an increase in cellular IP₃ (26), becoming available for regulation of NBCe1-B (21–23), NBCn1-A (24), CFTR (22, 23), and slc26a6 (27), thereby coordinating the activation of these transporters and epithelial fluid and HCO₃⁻ secretion (27).Cl⁻_in has not been previously considered as a signaling ion. Rather, Cl⁻ has heretofore mainly been viewed as an ion that is transported by various channels, coupled to Na⁺, K⁺, or HCO₃⁻ transport, and that plays a role regulating the plasma membrane potential (1, 7). Importantly, several studies have provided clues that Cl⁻_in may have regulatory and perhaps signaling functions. High Cl⁻_in was reported to inhibit the activity of the epithelial Na⁺ channel ENaC (28), the permeability of CFTR to HCO₃⁻ (29), TRPM7 activity (8), and perhaps the activity of the Na⁺/H⁺ exchanger NHE1 (30). Muscarinic stimulation of salivary gland acinar cells resulted in reduction in Cl⁻_in that is required for Na⁺ influx by the Na⁺/H⁺ exchanger and the Na⁺/K⁺/2Cl⁻ cotransporter (31).In the present study we asked whether Cl⁻_in functions as a signaling ion that modulates cellular Na⁺ and HCO₃⁻ concentrations through regulation of electrogenic NBC transporters. Our data indicate that Cl⁻_in regulates the function of NBCe1-B, NBCe2-C, and NBCe1-A via a cryptic Cl⁻ sites. In the basal state NBCe1-B is inhibited by Cl⁻_in interacting with a low affinity Cl⁻ site, whereas NBCe1-A is resistant to Cl⁻_in. By contrast, IRBIT-activated NBCe1-B is inhibited by low and high concentrations of Cl⁻_in due to interaction with high and low affinity Cl⁻_in motifs that depend on GXXXP motifs. Mutation of the G and P or of His in GXHXP in the autoinhibitory domain of NBCe1-B eliminated inhibition by low Cl⁻_in, whereas sparing inhibition of NBCe1-B by high Cl⁻_in. Mutation of a second GXXXP motif was required to eliminate inhibition by low affinity Cl⁻_in site. NBCe2-C is inhibited by a single high affinity GXXXP motif-dependent Cl⁻_in interacting site. Remarkably, deletion of the first 48 residues of NBCe1-A or of residues 29–41 uncovered inhibition of NBCe1-A by Cl⁻_in that was mediated by a GXXXP motif-dependent site homologous with the second GXXXP motif of NBCe1-B. These findings reveal a Cl⁻_in sensing mechanism that modulates the activity of NBCe1-B, NBCe2-C, and NBCe1-A. In transporting epithelia, such as the pancreatic and salivary ducts and the choroid plexus, we predict that a reduction in Cl⁻_in will dramatically increase transepithelial HCO₃⁻ transport and fluid secretion.     

Location of chloride and its possible functions in oxygen-evolving photosystem II revealed by X-ray crystallography

The chloride ion, Cl⁻, is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn₄Ca cluster. Its detailed location and function have not been identified, however. We substituted Cl⁻ with a bromide ion (Br⁻) or an iodide ion (I⁻) in PSII and analyzed the crystal structures of PSII with Br⁻ and I⁻ substitutions. Substitution of Cl⁻ with Br⁻ did not inhibit oxygen evolution, whereas substitution of Cl⁻ with I⁻ completely inhibited oxygen evolution, indicating the efficient replacement of Cl⁻ by I⁻. PSII with Br⁻ and I⁻ substitutions were crystallized, and their structures were analyzed. The results showed that there are 2 anion-binding sites in each PSII monomer; they are located on 2 sides of the Mn₄Ca cluster at equal distances from the metal cluster. Anion-binding site 1 is close to the main chain of D1-Glu-333, and site 2 is close to the main chain of CP43-Glu-354; these 2 residues are coordinated directly with the Mn₄Ca cluster. In addition, site 1 is located in the entrance of a proton exit channel. These results indicate that these 2 Cl⁻ anions are required to maintain the coordination structure of the Mn₄Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.     

Durability Properties of Ultra-High Performance Lightweight Concrete (UHPLC) with Expanded Glass

It is important to ensure the durability and safety of structures. In the case of newly developed materials that are outside the current rules, it is important to investigate all aspects of structural safety. The material studied in the following is a structural lightweight concrete with an ultra-high-performance matrix and expanded glass as a lightweight aggregate. The material, with a compressive strength of 60–100 MPa and a bulk density of 1.5–1.9 kg/dm³, showed high capillary porosities of 12 vol% (ultra-high-performance concretes (UHPC) < 5 vol%). Since the capillary porosity basically enables transport processes into the concrete, the material had to be examined more closely from the aspect of durability. Freeze-thaw resistance (68 g/m²) and chemical attack with sulfate at pH 3.5 for 12 weeks (16 g/m²) showed no increase in concrete corrosion. Targeted carbonation (0.53 mm/year^0.5) and chloride penetration resistance (6.0 × 10⁻¹³ to 12.6 × 10⁻¹³ m²/s) also showed good results against reinforcement corrosion. The results show that most of the measured capillary pores resulted from the lightweight aggregate and were not all present as a pore system. Thus, the durability was only slightly affected and the concrete can be compared to an UHPC. Only the abrasion resistance showed an increased value (22,000 mm³/5000 mm²), which, however, only matters if the material is used as a screed.     

A Comparative Study of the Properties of Recycled Concrete Prepared with Nano-SiO2 and CO2 Cured Recycled Coarse Aggregates Subjected to Aggressive Ions Environment

This research focused on the modification effects on recycled concrete (RC) prepared with nano-SiO₂ and CO₂ cured recycled coarse aggregates (RCA) subjected to an aggressive ions environment. For this purpose, RCA was first simply crushed and modified by nano-SiO₂ and CO_2, respectively, and the compressive strength, ions permeability as well as the macro properties and features of the interface transition zone (ITZ) of RC were investigated after soaking in 3.5% NaCl solution and 5% Na₂SO₄ solution for 30 days, respectively. The results show that nano-SiO₂ modified RC displays higher compressive strength and ions penetration resistance than that treated by carbonation. Besides, we find that ions attack has a significant influence on the microcracks width and micro-hardness of the ITZ between old aggregate and old mortar. The surface topography, elemental distribution and micro-hardness demonstrate that nano-SiO₂ curing can significantly decrease the microcracks width as well as Cl⁻ and SO₄²⁻ penetration in ITZ, thus increasing the micro-hardness, compared with CO₂ treatment.     

Evaluation of Eco-Efficient Concretes Produced with Fly Ash and Uncarbonated Recycled Aggregates

The fabrication of conventional concrete, as well as remains from demolition, has a high environmental impact. This paper assessed the eco-efficiency of concrete made with uncarbonated recycled concrete aggregates (RCA) and fly ash (FA). Two concrete series were produced with an effective water/cement ratio of 0.50 (Series 1) and 0.40 (Series 2). In both series, concretes were produced using 0% and 50% of RCA with 0%, 25% and 50% FA. After analysing the compressive strength, and carbonation and chloride resistance of those concretes, their eco-efficiency based on the binder intensity and CO₂-eq intensity was assessed. We found that the use of 50% uncarbonated RCA improved the properties of concretes produced with FA with respect to using natural aggregates. The concrete made of 25% FA plus RCA was considered the most eco-efficient based on the tests of compressive, carbonation and chloride properties with the values of 4.1 kg CO₂ m⁻³ MPa⁻¹, 76.3 kg CO₂ m⁻³ mm⁻¹ year^0.5 and 0.079 kg CO₂ m⁻³ C⁻¹, respectively. The uncarbonated RCA improved carbonation resistance, and FA improved chloride resistance. It can be concluded that the use of 50% un-carbonated RCA combined with FA considerably enhanced the properties of hardened concrete and their eco-efficiency with respect to concretes produced with natural aggregates.     

Effect of Cathodic Protection on Reinforced Concrete with Fly Ash Using Electrochemical Noise

Corrosion of steel reinforcement is the major factor that limits the durability and serviceability performance of reinforced concrete structures. Impressed current cathodic protection (ICCP) is a widely used method to protect steel reinforcements against corrosion. This research aimed to study the effect of cathodic protection on reinforced concrete with fly ash using electrochemical noise (EN). Two types of reinforced concrete mixtures were manufactured; 100% Ordinary Portland Cement (OCP) and replacing 15% of cement using fly ash (OCPFA). The specimens were under-designed protected conditions (−1000 ≤ E ≤ −850 mV vs. Ag/AgCl) and cathodic overprotection (E < −1000 mV vs. Ag/AgCl) by impressed current, and specimens concrete were immersed in a 3.5 wt.% sodium chloride (NaCl) Solution. The analysis of electrochemical noise-time series showed that the mixtures microstructure influenced the corrosion process. Transients of uniform corrosion were observed in the specimens elaborated with (OPC), unlike those elaborated with (OPCFA). This phenomenon marked the difference in the concrete matrix’s hydration products, preventing Cl⁻ ions flow and showing passive current and potential transients in most specimens.     

Adsorptive Removal of Phosphate from Aqueous Solutions Using Low-Cost Volcanic Rocks: Kinetics and Equilibrium Approaches

The contamination of surface and groundwater with phosphate originating from industrial and household wastewater remains a serious environmental issue in low-income countries. Herein, phosphate removal from aqueous solutions was studied using low-cost volcanic rocks such as pumice (VPum) and scoria (VSco), obtained from the Ethiopian Great Rift Valley. Batch adsorption experiments were conducted using phosphate solutions with concentrations of 0.5 to 25 mg·L⁻¹ to examine the adsorption kinetic as well as equilibrium conditions. The experimental adsorption data were tested by employing various equilibrium adsorption models, and the Freundlich and Dubinin-Radushkevich (D-R) isotherms best depicted the observations. The maximum phosphate adsorption capacities of VPum and VSco were calculated and found to be 294 mg·kg⁻¹ and 169 mg·kg⁻¹, respectively. A pseudo-second-order kinetic model best described the experimental data with a coefficient of correlation of R² > 0.99 for both VPum and VSco; however, VPum showed a slightly better selectivity for phosphate removal than VSco. The presence of competitive anions markedly reduced the removal efficiency of phosphate from the aqueous solution. The adsorptive removal of phosphate was affected by competitive anions in the order: HCO₃⁻ >F⁻ > SO₄⁻² > NO₃⁻ > Cl⁻ for VPum and HCO₃⁻ > F⁻ > Cl⁻ > SO₄⁻² > NO₃⁻ for VSco. The results indicate that the readily available volcanic rocks have a good adsorptive capacity for phosphate and shall be considered in future studies as test materials for phosphate removal from water in technical-scale experiments.     

From the Cover: PNAS Plus: Chloride transport-driven alveolar fluid secretion is a major contributor to cardiogenic lung edema

Alveolar fluid clearance driven by active epithelial Na⁺ and secondary Cl⁻ absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na⁺ channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl⁻ secretion and alveolar Cl⁻ influx were quantified by radionuclide tracing and alveolar Cl⁻ imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl⁻ secretion and alveolar Cl⁻ influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na⁺-K⁺-Cl⁻ cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR^−/− mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl⁻ secretion that were again CFTR-, NKCC-, and Na⁺-K⁺-ATPase–dependent. Our findings show a reversal of transepithelial Cl⁻ and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl⁻ and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.Traditionally, the formation of cardiogenic pulmonary edema has been attributed to passive fluid filtration across an intact alveolocapillary barrier along an increased hydrostatic pressure gradient. However, recent studies show that cardiogenic edema is critically regulated by active signaling processes. Activation of mechanosensitive endothelial ion channels increases lung vascular permeability (1), whereas alveolar epithelial cells lose their physiological ability to clear the distal airspaces from excess fluid by their capacity to actively transport ions across the epithelial barrier (2–4).In the intact lung, the predominant force driving alveolar fluid clearance is an active transepithelial Na⁺ transport from the alveolar into the interstitial space. A major portion of the apical Na⁺ entry is mediated by the amiloride-inhibitable epithelial Na⁺ channel (ENaC), with basolateral Na⁺ extrusion through the Na⁺-K⁺-ATPase (5). Cl⁻ and water are considered to follow paracellularly for electroneutrality and osmotic balance. In cardiogenic lung edema, the physiological protection against alveolar flooding provided by an intact alveolar fluid clearance is largely attenuated (3, 4). Previously, we have outlined the signaling events at the alveolocapillary barrier that underlie this inhibition of alveolar fluid clearance by showing that hydrostatic stress increases endothelial NO production in lung capillaries (6), which in turn, blocks alveolar Na⁺ and liquid absorption by a cGMP-dependent inhibition of epithelial ENaC (2).Unexpectedly, however, we observed that increased hydrostatic pressure not only blocks alveolar fluid clearance but reverses transepithelial fluid transport, resulting in effective alveolar fluid secretion that accounts for up to 70% of the total alveolar fluid influx at elevated hydrostatic pressure (2). This effect is not explicable by impaired alveolar fluid clearance and/or passive fluid leakage, and thus, it points to a previously unrecognized and potentially therapeutically exploitable pathomechanism in cardiogenic lung edema, namely alveolar fluid secretion driven by active transepithelial ion transport.Here, we aimed to analyze alveolar fluid secretion and its underlying cellular mechanisms in cardiogenic lung edema. We considered the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR) as a putative key ion channel in this scenario, because it permits bidirectional permeation of anions under physiologically relevant conditions (7). Hence, the direction of Cl⁻ flux by CFTR may reverse depending on actual electrochemical gradients, thus turning an absorptive into a secretory epithelium or vice versa. This notion is supported by reports describing CFTR as both an absorptive and secretory channel in the regulation of alveolar fluid homeostasis (8, 9). By a combination of indicator dilution, imaging, and radioactive tracer techniques for the measurement of alveolar ion and fluid fluxes in the isolated lung, we show a critical role for CFTR-mediated Cl⁻ secretion in cardiogenic lung edema and identify the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) as a therapeutic target in this pathology.     

Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl⁻) channel and a regulator of outwardly rectifying Cl⁻ channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl⁻ channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3–1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, ³⁶Cl⁻ efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl⁻ channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted α-helices 5 and 6, forms an essential part of the Cl⁻ channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl⁻ channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.     

The Synergistic Inhibitions of Tungstate and Molybdate Anions on Pitting Corrosion Initiation for Q235 Carbon Steel in Chloride Solution

In this work, the synergistic inhibitions of tungstate (WO₄²⁻) and molybdate (MoO₄²⁻) anions, including role and mechanism, on the initiation of pitting corrosion (PC) for Q235 carbon steel in chloride (Cl⁻) solution were investigated with electrochemical and surface techniques. The pitting potential (E_p) of the Q235 carbon steel in WO₄²⁻ + MoO₄^2- + Cl⁻ solution was more positive than that in WO₄²⁻ + Cl⁻ or MoO₄²⁻ + Cl⁻ solution; at each E_p, both peak potential and affected region of active pitting sites in WO₄²⁻ + MoO₄²⁻ + Cl⁻ solution were smaller than those in WO₄²⁻ + Cl⁻ or MoO₄²⁻ + Cl⁻ solution. WO₄²⁻ and MoO₄²⁻ showed a synergistic role to inhibit the PC initiation of the Q235 carbon steel in Cl⁻ solution, whose mechanism was mainly attributed to the influences of two anions on passive film. Besides iron oxides and iron hydroxides, the passive film of the Q235 carbon steel formed in WO₄²⁻ + Cl⁻, MoO₄²⁻ + Cl⁻, or WO₄²⁻ + MoO₄²⁻ + Cl⁻ solution was also composed of FeWO₄ plus Fe₂(WO₄)₃, Fe₂(MoO₄)₃, or Fe₂(WO₄)₃ plus Fe₂(MoO₄)₃, respectively. The film resistance and the defect quantity for Fe₂(WO₄)₃ plus Fe₂(MoO₄)₃ film were larger and smaller than those for FeWO₄ plus Fe₂(WO₄)₃ film and Fe₂(MoO₄)₃ film, respectively; for the inhibition of PC initiation, Fe₂(WO₄)₃ plus Fe₂(MoO₄)₃ film provided better corrosion resistance to Q235 carbon steel than FeWO₄ plus Fe₂(WO₄)₃ film and Fe₂(MoO₄)₃ film did.