Chloride Gradient Is Involved in Ammonium Influx in Human Erythrocytes.

The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl- and HCO3-). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled and lysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH4+-induced cell swelling and hemolysis, and the restoration of [Cl-] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs.

Rapid volume pulsation of the extracellular space coincides with epileptiform activity in mice and depends on the NBCe1 transporter.

KEY POINTS: Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3- cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may be a useful target in epilepsy patients who are resistant to current treatments. ​ ABSTRACT: The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (bicuculline methiodide model) and in vitro (hyaluronan synthase 3 knock-out, picrotoxin, bicuculline and 4-aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel aquaporin-4 (AQP4) and inwardly rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3- cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment.

VDAC1 as a target in cisplatin anti-tumor activity through promoting mitochondria fusion.

Cisplatin is one of the most effective anti-cancer drugs, but its efficacy is limited by the development of resistance. Previous studies have shown that mitochondria play critical roles in cisplatin cytotoxicity, however, the exact mechanism of mitochondria involved in cisplatin sensitivity has not been clarified. In this study, cisplatin triggered mitochondrial oxidative stress and the decrease of mitochondria membrane potential in human cervical cancer cells. Then we screened a series of mitochondrial relevant inhibitors, including mitochondrial mPTP inhibitors DIDS and CsA, and mitochondrial respiratory complex inhibitors Rot and TTFA. Among these, only DIDS, as the inhibitor of mitochondrial outer membrane protein VDAC1, showed strong antagonism against cisplatin toxicity. DIDS mitigated cisplatin-induced MFN1-dependent mitochondrial fusion, mitochondrial dysfunction and oxidative damage. These findings demonstrated that VDAC1 may serve as a potential therapeutic target in the increase sensitivity of cisplatin, which provides an attractive pharmacological therapy to improve the effectiveness of chemotherapy.

Toxicological analysis of stilbenes against the fall armyworm, Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a global pest of multiple economically important row crops and the development of resistance to commercially available insecticidal classes has inhibited FAW control. Thus, there is a need to identify chemical scaffolds that can provide inspiration for the development of novel insecticides for FAW management. This study aimed to assess the sensitivity of central neurons and susceptibility of FAW to chloride channel modulators to establish a platform for repurposing existing insecticides or designing new chemicals capable of controlling FAW. Potency of select chloride channel modulators were initially studied against FAW central neuron firing rate and rank order of potency was determined to be fipronil > lindane > Z-stilbene > DIDS > GABA > E-stilbene. Toxicity bioassays identified fipronil and lindane as the two most toxic modulators studied with topical LD50's of 41 and 75 ng/mg of caterpillar, respectively. Interestingly, Z-stilbene was toxic at 300 ng/mg of caterpillar, but no toxicity was observed with DIDS or E-stilbene. The significant shift in potency between stilbene isomers indicates structure-activity relationships between stilbene chemistry and the binding site in FAW may exist. The data presented in this study defines the potency of select chloride channel modulators to FAW neural activity and survivorship to establish a platform for development of novel chemical agents to control FAW populations. Although stilbenes may hold promise for insecticide development, the low toxicity of the scaffolds tested in this study dampen enthusiasm for their development into FAW specific insecticides.

Cryo-EM structures of DrNKCC1 and hKCC1: a new milestone in the physiology of cation-chloride cotransporters.

New milestones have been reached in the field of cation-Cl- cotransporters with the recently released cryo-electron microscopy (EM) structures of the Danio rerio (zebrafish) Na+-K+-2Cl- cotransporter (DrNKCC1) and the human K+-Cl- cotransporter (hKCC1). In this review we provide a brief timeline that identifies the multiple breakthroughs in the field of solute carrier 12 transporters that led to the structure resolution of two of its key members. While cation-Cl- cotransporters share the overall architecture of carriers belonging to the amino acid-polyamine-organocation (APC) superfamily and some of their substrate binding sites, several new insights are gained from the two individual structures. A first major feature relates to the largest extracellular domain between transmembrane domain (TMD) 5 and TMD6 of KCC1, which stabilizes the dimer and forms a cap that likely participates in extracellular gating. A second feature is the conservation of the K+ and Cl- binding sites in both structures and evidence of an unexpected second Cl- coordination site in the KCC1 structure. Structural data are discussed in the context of previously published studies that examined the basic and kinetics properties of these cotransport mechanisms. A third characteristic is the evidence of an extracellular gate formed by conserved salt bridges between charged residues located toward the end of TMD3 and TMD4 in both transporters and the existence of an additional neighboring bridge in the hKCC1 structure. A fourth feature of these newly solved structures relates to the multiple points of contacts between the monomer forming the cotransporter homodimer units. These involve the TMDs, the COOH-terminal domains, and the large extracellular loop for hKCC1.

4,4'-Diisothiocyanato-2,2'-Stilbenedisulfonic Acid (DIDS) Modulates the Activity of KCNQ1/KCNE1 Channels by an Interaction with the Central Pore Region.

BACKGROUND/AIMS: The cardiac current IKs is carried by the KCNQ1/KCNE1-channel complex. Genetic aberrations that affect the activity of KCNQ1/KCNE1 can lead to the Long QT Syndrome 1 and 5 and, thereby, to a predisposition to sudden cardiac death. This might be prevented by pharmacological modulation of KCNQ1/KCNE1. The prototypic KCNQ1/KCNE1 activator 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) represents a candidate drug. Here, we study the mechanism of DIDS action on KCNQ1/KCNE1. METHODS: Channels were expressed in Xenopus oocytes and iPSC cardiomyocytes. The role of the central S6 region was investigated by alanin-screening of KCNQ1 residues 333-338. DIDS effects were measured by TEVC and MEA. RESULTS: DIDS-action is influenced by the presence of KCNE1 but not by KCNQ1/KCNE1 stochiometry. V334A produces a significant higher increase in current amplitude, whereas deactivation (slowdown) DIDS-sensitivity is affected by residues 334-338. CONCLUSION: We show that the central S6 region serves as a hub for allosteric channel activation by the drug and that DIDS shortens the pseudo QT interval in iPSC cardiomyocytes. The elucidation of the structural and mechanistic underpinnings of the DIDS action on KCNQ1/KCNE1 might allow for a targeted design of DIDS derivatives with improved potency and selectivity.

The teleost fish intestine is a major oxalate-secreting epithelium.

Oxalate is a common constituent of kidney stones, but the mechanism of its transport across epithelia is not well understood. With prior research on the role of the intestine focused on mammals, the present study considered oxalate handling by teleost fish. Given the osmotic challenge of seawater (SW), marine teleosts have limited scope for urinary oxalate excretion relative to freshwater (FW) taxa. The marine teleost intestine was hypothesized as the principal route for oxalate elimination, thus demanding epithelial secretion. To test this, intestinal 14C-oxalate flux was compared between FW- and SW-acclimated sailfin molly (Poecilia latipinna). In SW, oxalate was secreted at remarkable rates (367.90±22.95 pmol cm-2 h-1), which were similar following FW transfer (387.59±27.82 pmol cm-2 h-1), implying no regulation by salinity. Nevertheless, this ability to secrete oxalate at rates 15-19 times higher than the mammalian small intestine supports this proposal of the teleost gut as a major, previously unrecognized excretory pathway.

Isothiocyanate groups of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibit cell penetration of octa-arginine (R8)-fused peptides.

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine-a well-known cell-penetrating peptide (CPP)-is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide-ie, mitochondrial targeting domain (MTD)-to the cytosol. We found that 4,4'-diisothiocyanatostilbene-2,2'-di-sulfonate (DIDS)-an anion channel blocker-inhibited the ability of octa-arginine (R8)-fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di-sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di-sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules.

Voltage-gated chloride channel blocker DIDS as an acaricide for Varroa mites.

The Varroa mite is a primary driver behind periodical losses of honey bee colonies. These mites require honey bees for food and reproduction and, in turn, elicit physiological deficiencies and diseases that compromise colony health. Current acaricides for Varroa mite control, such as Apistan® (the pyrethroid tau-fluvalinate), CheckMite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) target the nervous system, can have adverse health effects on honey bees, and have limited effectiveness due to reported resistance issues. New target sites are needed to circumvent these obstacles in Varroa mite management, and voltage-gated chloride channels (VGCCs) are promising candidates due to their important role in the maintenance of nerve and muscle excitability in arthropod pests. Toxicological analysis of Varroa mites sensitive to tau-fluvalinate and coumaphos and Varroa mites with reduced sensitivity to these acaricides showed a significant increase in metabolic detoxification enzyme activities for the latter. Acetylcholinesterase activity in the Varroa mites exhibiting reduced mortality to coumaphos was significantly less sensitive to coumaphos-oxon compared to coumaphos-sensitive Varroa mites, which suggests target-site insensitivity to the acaricide. Voltage-gated chloride channel blocker DIDS had significantly greater field efficacy compared to Apistan® and CheckMite+® against Varroa mites from honey bee hives where tau-fluvalinate and coumaphos were observed to be ineffective, respectively. These data suggest that DIDS, and potentially other stilbene chemistries, might serve as candidates for continued field efficacy testing of alternative acaricides in apiaries where Apistan®- and CheckMite+® efficacy has been. reduced or lost for Varroa mites.

4,4'-Diisothiocyanatostilbene-2,2'-disulfonate modulates voltage-gated K+ current and influences cell cycle arrest in androgen sensitive and insensitive human prostate cancer cell lines.

The stilbene derivative, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), an anion channel blocker is used in the present study to evaluate its modulatory effect on voltage-gated K+ current (IK) in human prostate cancer cell lines (LNCaP and PC-3). Voltage-gated K+ (KV) channels in the plasma membrane are critically involved in the proliferation of tumor cells. Therefore, KV channels are considered as a novel potential target for cancer treatment. The results of the present study show that the external perfusion of DIDS activates IK in a concentration-dependent manner, although the known K+ channel blocker TEA failed to block the DIDS activated IK in PC-3 cells. Whereas, in LNCaP cells, the higher concentration of DIDS blocked IK, though this effect was not completely recovered after washout. The difference in function of DIDS might be due to the expression of different Kv channel isoforms in LNCaP and PC-3 cells. Further, the anticancer studies show that treatment of DIDS significantly induced G2/M phase cell cycle arrest and induced moderate and low level of cell death in LNCaP and PC-3 cells respectively. This finding reveals that DIDS modulates IK and exerts cell cycle arrest and cell death in LNCaP and PC-3 cells.

A peptide containing Noxa mitochondrial-targeting domain induces cell death via mitochondrial and endoplasmic reticulum disruption.

Noxa is a weak apoptosis activator consisting of a BH3 domain and a mitochondrial-targeting domain (MTD). BH3 binds Mcl-1 and Bcl2A1 and inactivates their anti-apoptotic activities, while MTD delivers BH3 to mitochondria. Previously we revealed that MTD may also function as an inducer of necrosis via conjugation with octa-arginine, which induces cytosolic Ca2+ influx from mitochondria. However, the mechanism(s) underlying this process has not been elucidated yet. Here, we show that calcium influx induced by an MTD peptide fused with octa-arginine residue (R8:MTD) originates not only from mitochondria but also from the extracellular space. However, calcium spikes were not sufficient for necrosis. R8:MTD induced mitochondrial permeability transition pore opening, fragmentation, and swelling. These mitochondrial events induced by MTD appeared to be necessary for necrosis induction, since DIDS, a VDAC inhibitor, inhibited the mitochondrial swelling and cell death induced by MTD. We show that R8:MTD disrupted endoplasmic reticulum (ER) structures but not peroxisomes or Golgi, indicating that R8:MTD causes necrosis by inducing ER events as well.

Inhibition of VDAC1 Protects Against Glutamate-Induced Oxytosis and Mitochondrial Fragmentation in Hippocampal HT22 Cells.

The involvement of glutamate in neuronal cell death in neurodegenerative diseases and neurotrauma is mediated through excitotoxicity or oxytosis. The latter process induces oxidative stress via glutamate-mediated inhibition of cysteine transporter xCT, leading to depletion of the cellular glutathione pool. Mitochondrial damage, loss of mitochondrial membrane potential (MMP), and depletion of energy metabolites have been shown in this process. The Voltage-Dependent Anion Channel-1 (VDAC1) is one of the main components of the mitochondrial outer membrane and plays a gatekeeping role in mitochondria-cytoplasm transport of metabolites. In this study, we explored the possible participation of VDAC-1 in the pathophysiology of oxytosis. Administration of glutamate in HT22 cells that lack the glutamate ionotropic receptors induced an upregulation and oligomerization of VDAC1. This was associated with an increase in ROS and loss of cell survival. Glutamate-mediated oxytosis in this model also decreased MMP and promoted ATP depletion, resulting in translocation of cytochrome c (cyt C) and apoptosis inducing factor (AIF) from mitochondria into the cytosol. This was also accompanied by cleavage of AIF to form truncated AIF. Inhibition of VDAC1 oligomerization using 4,4'-Diisothiocyanatostilbene-2,2'-disulfonate (DIDS), significantly improved the cell survival, decreased the ROS levels, improved mitochondrial functions, and decreased the mitochondrial damage. Notably, DIDS also inhibited the mitochondrial fragmentation caused by glutamate, indicating the active role of VDAC1 oligomerization in the process of mitochondrial fragmentation in oxytosis. These results suggest a critical role for VDAC1 in mitochondrial fragmentation and its potential therapeutic value against glutamate-mediated oxidative neurotoxicity.

Thermal sensitivity and haemolysis of erythrocytes with membranopathy.

Measuring the impedance of heated suspensions of erythrocytes and erythrocyte ghost membranes, two thermally-induced alterations are registered in the plasma membrane at TA (denaturation of spectrin with inducing temperature at 49,5 °C) and TG (hyperthermic activation of basal ion permeability with inducing temperature at 60.7 °C). In this study erythrocytes from 9 healthy patients and 15 patients with hemolytic anemia were studied and divided into four groups depending on their TA and TG top temperatures. The TA and TG of erythrocytes with hemoglobinopathy were the same as those of control erythrocytes while those of erythrocytes with membranopathy were significantly reduced. In erythrocytes with severe membranopathy, the TG was decreased by about 5 °C. In latter cells the normal value of TG was restored and the resistance to thermal haemolysis was increased by 90% after the specific stabilization of band 3 protein by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). Obtained results indicate the involvement of band 3 in the membrane alteration at TG and in the heat target responsible for thermal haemolysis.

DIDS inhibits overexpression BAK1-induced mitochondrial apoptosis through GSK3ß/ß-catenin signaling pathway.

Bcl-2 homologous antagonist/killer (BAK1) is a critical regulator of mitochondrial apoptosis. Although upregulation of BAK1 induces apoptosis has been established, the underlying molecular mechanism is far from clear. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an organic anion used as a blocker of anion exchangers and chloride channels, has been proved to rescue cell apoptosis both in vitro and in vivo. However, whether DIDS can inhibit BAK1-induced mitochondrial apoptosis remains undefined. Thus, this study aimed to explore whether DIDS could protect BAK1-induced apoptosis through GSK3ß/ß-catenin signaling pathway. The results showed overexpression BAK1 in 293T cells induced mitochondrial apoptosis accompanied by increasing the expression levels of cleaved caspase-9, -3, poly (ADP-ribose) polymerase (PARP) and reducing the MMP. Furthermore, overexpression BAK1 decreased the expression levels of Ser9-GSK3ß and ß-catenin. In addition, lithium chloride (LiCl), an activator of Wnt/ß-catenin signaling pathway, markedly attenuated overexpression BAK1-induced mitochondrial apoptosis by restoring the expression levels of Ser9-GSK3ß and ß-catenin. Finally, DIDS absolutely abolished overexpression BAK1-mediated mitochondrial apoptosis through recovering the expression levels of Ser9-GSK3ß and ß-catenin. Taken together, our results reveal that DIDS blocks overexpression BAK1-induced mitochondrial apoptosis through GSK3ß/ß-catenin pathway.

A Potassium-Selective Current Affected by Micromolar Concentrations of Anion Transport Inhibitors.

BACKGROUND/AIMS: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. METHODS: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). RESULTS: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. CONCLUSION: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.

Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line.

The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC50 of 5.1µM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC50=116nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening.

Al(3+) -promoted fluoride accumulation in tea plants (Camellia sinensis) was inhibited by an anion channel inhibitor DIDS.

BACKGROUND: Generally, tea plants are grown in acid soil which is rich in aluminum (Al) and fluoride (F). A recent publication showed that pretreatment with Al(3+) promoted F accumulation in tea plants by increasing endogenous Ca(2+) and calmodulin (CaM). A high level of F in tea leaves not only impairs tea quality but also might pose a health risk for people drinking tea regularly. Therefore it is important to try to find some clues which might be beneficial in controlling F accumulation in tea plants grown in acid soil (Al(3+) ). RESULTS: It was found that diisothiocyanostilbene-2,2-disulfonic acid (DIDS) significantly reduced Al(3+) -promoted F accumulation in tea plants. Additionally, Al(3+) plus DIDS treatment stimulated significantly higher Ca(2+) efflux and decreased the CaM level in tea roots compared with Al(3+) treatment. Besides, significantly higher depolarization of membrane potential was shown in tea roots treated with Al(3+) plus DIDS than in those treated with Al(3+) , as well as higher net total H(+) efflux and plasma membrane H(+) -ATPase activity. CONCLUSION: Al(3+) -promoted F accumulation in tea plants was inhibited by an anion channel inhibitor DIDS. Ca(2+) /CaM and membrane potential depolarization may be the components involved in this process. © 2016 Society of Chemical Industry.

Human SLC4A11-C functions as a DIDS-stimulatable H⁺(OH⁻) permeation pathway: partial correction of R109H mutant transport.

The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H(+)(OH(-)) permeation pathway. Disulfonic stilbenes, including 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H(+)(OH(-)) flux by 150-200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4'-diaminostilbene-2,2'-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H(+)(OH(-)) flux through the mutant transporter by ∼40-90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.

Pathological impact of hyperpolarization-activated chloride current peculiar to rat pulmonary vein cardiomyocytes.

Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs(+) resistant, and was not affected by removal of external Na(+) or K(+). The current was inhibited with Cd(2+), and the reversal potential was sensitive to changes in [Cl(-)] on either side of the membrane in a manner consistent with a Cl(-) selective channel. Cl(-) channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl(-) current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl(-) current in rat PVs was shifted to negative potentials as [Cl(-)]i increased, (ii) the Cl(-) current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl(-) channel, and that the channel may play a functional role in norepinephrine-induced automaticity.

Characterization of the mouse ClC-K1/Barttin chloride channel.

Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(-)>Br(-)>NO3(-)>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).