Opening Of Chloride Channels Research Articles

Chromatographic methods for the determination of various barbiturates: A review

Barbiturates are generally used in the treatment of epilepsy, convulsions, and anesthesia. Different analytical methods are used to identify, quantify, and determine barbiturates in other dosage forms. The cells of the central nervous system are highly sensitive to barbiturates. Barbiturates will enhance the GABA receptor activity and result in the inhibitory action of neurotransmitters due to the opening of chloride channels. This review summarizes the analysis of Barbiturates in Biological samples like hair, blood, serum, urine, and pharmaceutical dosage forms. Bioanalytical techniques have been used to analyze these drugs in biological samples such as blood, urine, and hair. These analytical techniques include High-performance liquid chromatography (HPLC), Gas chromatography (GC), Mass spectroscopy (MS), Thin-layer chromatography (TLC), and Ultra-violet spectroscopy (UV), Capillary electrophoresis which can produce the most appropriate results. This review gathers information, for the first time, regarding the determination of Secobarbital, Amobarbital, Pentobarbital, Mephobarbital, and Pentobarbital analytical methods for these drugs.

Insights into CLC-0's Slow-Gating from Intracellular Proton Inhibition.

The opening of the Torpedo CLC-0 chloride (Cl-) channel is known to be regulated by two gating mechanisms: fast gating and slow (common) gating. The structural basis underlying the fast-gating mechanism is better understood than that of the slow-gating mechanism, which is still largely a mystery. Our previous study on the intracellular proton (H+i)-induced inhibition of the CLC-0 anionic current led to the conclusion that the inhibition results from the slow-gate closure (also called inactivation). The conclusion was made based on substantial evidence such as a large temperature dependence of the H+i inhibition similar to that of the channel inactivation, a resistance to the H+i inhibition in the inactivation-suppressed C212S mutant, and a similar voltage dependence between the current recovery from the H+i inhibition and the recovery from the channel inactivation. In this work, we further examine the mechanism of the H+i inhibition of wild-type CLC-0 and several mutants. We observe that an anion efflux through the pore of CLC-0 accelerates the recovery from the H+i-induced inhibition, a process corresponding to the slow-gate opening. Furthermore, various inactivation-suppressed mutants exhibit different current recovery kinetics, suggesting the existence of multiple inactivated states (namely, slow-gate closed states). We speculate that protonation of the pore of CLC-0 increases the binding affinity of permeant anions in the pore, thereby generating a pore blockage of ion flow as the first step of inactivation. Subsequent complex protein conformational changes further transition the CLC-0 channel to deeper inactivated states.

Zerumbone, a major sesquiterpene from Syringa pinnatifolia Hemsl., exerts the sedative effect by regulating GABAergic nervous system

Ethnopharmacological relevanceZerumbone (ZER) is a humulane sesquiterpenoid isolated from Syringa pinnatifolia Hemsl. (SP), its content accounts for 64.7% of volatile oil and 0.86% of total ethanol extract (TEE), representing one of characteristic ingredient of SP. As a representative Mongolian medicine with anti-“Khii”, anti-asthma, and clearing-heat effects, SP has been used for the treatment of cardiovascular diseases, upset, insomnia, and other symptoms. Aim of studyPrevious results showed that TEE has sedative effect, but the pharmacological substances and its sedative mechanism remains unclear. This study aims to determine whether ZER, as one of major and characteristic sesquiterpenoids of SP, contributes to the sedative effect of SP and its underlying mechanism. Materials and methodsLocomotor activity and threshold dose of pentobarbital sodium sleep experiments were used to evaluate the sedative effects in mice. ELISA assay was used to examine the level of GABA/Glu ratio in rats hippocampus, cortex and hypothalamus tissue. The binding ability of ZER with glutamic acid decarboxylase 67 (GAD67) and Gephyrin protein were predicted by molecular docking. Western blot and Immunohistochemistry assay were used to determine the expression of GABAergic nerve system related proteins (GAD67, Gephyrin) in rat’s hypothalamus. ZER was co-administrated with flumazenil and bicuculline (GABAA antagonist) to determine whether it acts on GABAA receptor. Furthermore, MQAE assay was used to test the effect of ZER on the chloride ion concentration in cerebellar granule cells. ResultsCurrent data demonstrated that ZER dose-dependently (5–20 mg/kg) reduces the locomotor activity and sleep latency of mice, and extend sleeping time of mice. The results of ELISA showed that ZER increases the level of GABA/Glu in rats brain tissue, in particular in hypothalamus. Molecular docking results revealed that ZER has a strong affinity to GAD67 and Gephyrin protein. The Western blot and Immunohistochemistry data indicated that ZER up-regulates the expression of GAD67 and Gephyrin protein in rat’s hypothalamus. Antagonism test results demonstrated that flumazenil and bicuculline reverse the effect of ZER on threshold dose of pentobarbital sodium sleep experiments. In addition, ZER also could dose-dependently (5–20 μM) increase the chloride ion concentration in cerebellar granule cell, suggesting that ZER induces the opening of chloride channel, exerts central inhibitory effect. ConclusionZER has a significant sedative effect in mice and rat, and the effect is associated with GABAergic nervous system. The present results suggest that ZER, as one of the major bioactive ingredients of SP, contributes to the sedative effect and provide substantial evidence for its traditional use of anti-“Khii” in clinic of Syringa pinnatifolia.

Investigating the Role of GABA in Neural Development and Disease Using Mice Lacking GAD67 or VGAT Genes.

Normal development and function of the central nervous system involves a balance between excitatory and inhibitory neurotransmission. Activity of both excitatory and inhibitory neurons is modulated by inhibitory signalling of the GABAergic and glycinergic systems. Mechanisms that regulate formation, maturation, refinement, and maintenance of inhibitory synapses are established in early life. Deviations from ideal excitatory and inhibitory balance, such as down-regulated inhibition, are linked with many neurological diseases, including epilepsy, schizophrenia, anxiety, and autism spectrum disorders. In the mammalian forebrain, GABA is the primary inhibitory neurotransmitter, binding to GABA receptors, opening chloride channels and hyperpolarizing the cell. We review the involvement of down-regulated inhibitory signalling in neurological disorders, possible mechanisms for disease progression, and targets for therapeutic intervention. We conclude that transgenic models of disrupted inhibitory signalling—in GAD67+/− and VGAT−/− mice—are useful for investigating the effects of down-regulated inhibitory signalling in a range of neurological diseases.

Interleukin 1 beta-induced chloride currents are important in osteoarthritis onset: an in vitro study

Persistent hypotonic and inflammatory conditions in the joint cavity can lead to the loss of cartilage matrix and cell death, which are the important mechanisms of osteoarthritis (OA) onset. Previous studies have confirmed that the existence of a hypotonic environment is a red flag for inflammation, as hypotonic environment induces the opening of the chloride channel of the cell and promotes chloride ion efflux, which prompts the cell volume to increase. Chloride channels play an important role in the regulation of mineralization and chondrocyte death. Here, we reported that OA chondrocytes showed a significant increase of cell death rate and the imbalance of cartilage matrix catabolism. We found that the distribution of skeleton protein F-actin was disordered. In addition, the volume-sensitive chloride current of OA chondrocytes decreased significantly with the increase of the expression levels of inflammation-related proteins caspase-1, caspase-3, and NLRP3. Moreover, interleukin-1β (IL-1β) showed a potential to activate the chloride current of normal chondrocytes. These results indicate that IL-1β-induced chloride channel opening in chondrocytes may be closely related to the occurrence of OA. This chloride channel opening process may therefore be a potential target for the treatment of OA.

Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR

BackgroundLubiprostone (LBP) is a novel chloride channel opener that has been reported to activate chloride channel protein 2 (ClC-2) and cystic fibrosis transmembrane conductance regulator (CFTR). LBP facilitates fluid secretion by activating CFTR in the intestine and is used as a drug for treating chronic constipation. While ClC-2 and CFTR expression has been confirmed in cardiomyocytes (CMs), the effect of LBP on CMs has not yet been investigated. Thus, the present study aimed to investigate the effect of LBP on CMs using mouse-induced pluripotent stem (iPS) cell-derived CMs (iPS-CMs).MethodsWe induced mouse iPS cells into CMs through embryoid body (EB) formation. We compared the differentiated cells to CMs isolated from adult and fetal mice using gene expression, spontaneous beating rate, and contraction ratio analyses.ResultsGene expression analysis revealed that, in the iPS-CMs, the mRNA expression of the undifferentiated cell markers Rex1 and Nanog decreased, whereas the expression of the unique cardiomyocyte markers cardiac troponin I (cTnI) and cardiac troponin T (cTNT), increased. Immunostaining showed that the localization of cTnI and connexin-43 in the iPS-CMs was similar to that in the primary fetal CMs (FCMs) and adult CMs (ACMs). LBP decreased the spontaneous beating rate of the iPS-CMs and FCMs, and decreased the contraction ratio of the iPS-CMs and ACMs. The reduction in the beating rate and contraction ratio caused by LBP was inhibited by glycine hydrazide (GlyH), which is a CFTR inhibitor.ConclusionThese results suggest that LBP stimulates CFTR in CMs and that LBP has negative chronotropic and inotropic effects on CMs. LBP may be useful for treating cardiac diseases such as heart failure, ischemia, and arrhythmia.

Effects of Ivermectin Exposure on Regeneration of D. dorotocephala Planaria: Exploiting Human-Approved Ion Channel Drugs as Morphoceuticals.

Transformative applications in regenerative medicine await increased control of processes implementing repair and remodeling of complex living structures. Recent work reveals ion channel drugs as a powerful toolkit for modulating endogenous bioelectric circuits that control growth and form in vivo and in vitro. It is therefore especially important to develop assays in model systems that will enable the testing of these "morphoceuticals"-compounds with predictable effects on anatomical structure. The regenerative planaria are an ideal model system for this purpose. Several studies have shown a role for bioelectric signaling in planarian regeneration, but these have focused on Dugesia japonica and Schmidtea mediterranea. It is not known how the alterations of ion channel activity would affect regeneration in other species of planaria-an important aspect of building robust computational models of bioelectric circuits. Here, the effect of ivermectin (IVM), a chloride channel opener drug commonly used to combat heartworm is tested, on regeneration in a new species of planaria: Dugesia dorotocephala. Exposure to IVM during regeneration results in patterning abnormalities, such as bifurcated tails with partial heads, as well as delayed regeneration. These data extend our understanding of the effects of human-approved ion channel drugs on regenerative processes.

The potent insulin secretagogue effect of betulinic acid is mediated by potassium and chloride channels

Betulinic acid (BA) has been described as an insulin secretagogue which may explain its potent antihyperglycemic effect; however, the exact role of BA as an insulinogenic agent is not clear. The aim of this study was to investigate the mechanism of BA on calcium influx and static insulin secretion in pancreatic islets isolated from euglycemic rats. We found that BA triggers calcium influx by a mechanism dependent on ATP-dependent potassium channels and L-type voltage-dependent calcium channels. Additionally, the voltage-dependent and calcium-dependent chloride channels are also involved in the mechanism of BA, probably due to an indirect stimulation of calcium entry and increased intracellular calcium. Additionally, the downstream activation of PKC, which is necessary for the effect of BA on calcium influx, is involved in the full stimulatory response of the triterpene. BA stimulated the static secretion of insulin in pancreatic islets, indicating that the abrupt calcium influx may be a key step in its secretagogue effect. As such, BA stimulates insulin secretion through the activation of electrophysiological mechanisms, such as the closure of potassium channels and opening of calcium and chloride channels, inducing cellular depolarization associated with metabolic-biochemical effects, in turn activating PKC and ensuring the secretion of insulin.

1,25-Dihydroxycholecalciferol (calcitriol) modifies uptake and release of 25-hydroxycholecalciferol in skeletal muscle cells in culture

The major circulating metabolite of vitamin D3, 25-hydroxycholecalciferol [25(OH)D], has a remarkably long half-life in blood for a (seco)steroid. Data from our studies and others are consistent with the hypothesis that there is a role for skeletal muscle in the maintenance of vitamin D status. Muscle cells internalise vitamin D-binding protein (DBP) from the circulation by means of a megalin/cubilin plasma membrane transport mechanism. The internalised DBP molecules then bind to actin and thus provide an intracellular array of high affinity binding sites for its specific ligand, 25(OH)D. There is evidence that the residence time for DBP in muscle cells is short and that it undergoes proteolytic degradation, releasing bound 25(OH)D. The processes of internalisation of DBP and its intracellular residence time, bound to actin, appear to be regulated. To explore whether 1,25-dihydroxycholecalciferol (calcitriol) has any effect on this process, cell cultures of myotubes and primary skeletal muscle fibers were incubated in a medium containing 10−10M calcitriol but with no added DBP. After 3h pre-incubation with calcitriol, the net uptake of 25(OH)D by these calcitriol-treated cells over a further 4h was significantly greater than that in vehicle-treated control cells. This was accompanied by a significant increase in intracellular DBP protein. However, after 16h of pre-incubation with calcitriol, the muscle cells showed a significantly depressed ability to accumulate 25(OH)D compared to control cells over a further 4 or 16hours. These effects of pre-incubation with calcitriol were abolished in fibers from VDR-knockout mice. The effect was also abolished by the addition of 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride channel opening. Incubation of C2 myotubes with calcitriol also significantly reduced retention of previously accumulated 25(OH)D after 4 or 8h. It is concluded from these in vitro studies that calcitriol can modify the DBP-dependent uptake and release of 25(OH)D by skeletal muscle cells in a manner that suggests some inducible change in the function of these cells.

Effect of propofol on phosphatidyl inositol 3-serine kinase/protein kinase signaling pathway in breast tumor cells

Objective To study the effect of propofol on phosphatidyl inositol 3-serine kinase/protein kinase (PI3K/AKT) signaling pathway mediated by γ-aminobutyric acid receptors on breast cancer cell michigan cancer foundation-7 (MCF-7 cell) surface and to understand whether it presents inhibitory effect on proliferation of breast cancer cells. Methods According to the concentration of propofol, it is classified into propofol groups of 100 μmol/L, 300 μmol/L, 1 mmol/L, as well as 3 mmol/L. Set up blank control group without the intervention of propofol and intervention groups with different concentrations of propofol + γ-aminobutyric acid antagonist bicuculline. Apply whole-cell patch clamp technique to observe the effect of different concentrations of propofol in the intervention groups, control group as well as propofol combined with γ-aminobutyric acid antagonist group on chloride channels on the surface of MCF-7 cell membrane. Use protein signet ring method to detect the expression of Akt protein in the PI3K/Akt signaling path within MCF-7 cells. Use methyl thiazol tetrazolium (MTT) method to determine the effect of propofol on MCF-7 cell viability; compared both of them. Results (1) With the increase of cell membrane potential, propofol shall concentration-dependently simulate the chlorine ion current intensity in MCF-7 cells, with chlorine ion current intensity in MCF-7 cells of 3 mmol/L propofol group showing the intensity was (40.13±9.21) Pa, the control group showing the intensity was (19.46±8.53) Pa, the maximum difference compared to the control group, and the difference was statistically significant (P=0.032); after adding γ-aminobutyric acid antagonist bicuculline, the effect of propofol on the chlorine ion current intensity in MCF-7 cells weakened, but it had no significant difference with the 3mmol/L propofol group (16.92±7.47) Pa and the control group (18.61±8.19) Pa, (P=0.063); (2) Protein signet ring method showed that propofol has inhibitory effect in Akt protein expression in PI3K/Akt signaling pathway of MCF-7 cells; (3) The inhibitory effect of propofol on the survival of MCF-7 cells shall increase with the concentration of propofol. (4) The immunohistochemistry results showed that, breast cancer MCF-7 cells in propofol group was significantly lower than that of the control group when exposing tumor tissue to propofol for 72 h. Conclusion By stimulating the opening of chloride channel in MCF-7 cells, propofol of high concentration shall intensify its interation with γ-aminobutyric acid receptors in breast cancer cell surface to inhibit the activity of PI3K/Akt signaling pathway, and ultimately inhibit breast cancer cell proliferation. Key words: Propofol; Breast cancer cells; Phosphatidyl inositol 3-serine kinase/protein kinase signaling pathway; Cell proliferation

Evaluation of antiangiogenic potential of lubiprostone, a clc-2 channel agonist by chorioallantoic Membrane assay. (cam)

Angiogenesis is the formation of new blood vessels. Abundant ion channels are located on the endothelial cell surface. Ion channels have significant role in cell proliferation and thus in vascular angiogenesis. The antiangiogenic activity of a specific voltage gated Chloride channel opener ClC2 (Lubiprostone) was evaluated by Chorioallantoic Membrane (CAM) Assay (in ovo).The standard antiangiogenic drug used for comparison was Bevacizumab. All the data obtained from the experimental groups have been compared to the control group. The data was analyzed by one-way ANOVA followed by post hoc test, the Dunnett's test to compare mean of every group with the control mean, using graph pad prism5.0 software. In the CAM assay, the parameters evaluated were number of branches per unit area and angiogenic score. Medium (Lubiprostone 10 -5 M /egg) and high (Lubiprostone 10 -4 M /egg) doses of both the channel blockers gave significant results. From the results obtained, Lubiprostone has effective anti-angiogenesis which may be therapeutically beneficial .The molecular pathways may be due to negative regulation of cell volume, cell migration and thus proliferation.

Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.

Cystic fibrosis (CF) is a lethal monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that entails the (diagnostic) increase in sweat electrolyte concentrations, progressive lung disease with chronic inflammation and recurrent bacterial infections, pancreatic insufficiency, and male infertility. Therapies aimed at restoring the CFTR defect have emerged. Thus, a small molecule which facilitates chloride channel opening, the potentiator Ivacaftor, has been approved for the treatment of CF patients bearing a particular class of rare CFTR mutations. However, small molecules that directly target the most common misfolded CFTR mutant, F508del, and improve its intracellular trafficking in vitro, have been less effective than expected when tested in CF patients, even in combination with Ivacaftor. Thus, new strategies are required to circumvent the F508del-CFTR defect. Airway and intestinal epithelial cells from CF patients bearing the F508del-CFTR mutation exhibit an impressive derangement of cellular proteostasis, with oxidative stress, overactivation of the tissue transglutaminase (TG2), and disabled autophagy. Proteostasis regulators such as cysteamine can rescue and stabilize a functional F508del-CFTR protein through suppressing TG2 activation and restoring autophagy in vivo in F508del-CFTR homozygous mice, in vitro in CF patient-derived cell lines, ex vivo in freshly collected primary patient’s nasal cells, as well as in a pilot clinical trial involving homozygous F508del-CFTR patients. Here, we discuss how the therapeutic normalization of defective proteostasis can be harnessed for the treatment of CF patients with the F508del-CFTR mutation.

The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells

Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl− currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl− current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl− current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl− current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl− current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels.

The Role of Ivacaftor in Severe Cystic Fibrosis in a Patient With the R117H Mutation

Cystic fibrosis (CF) conductance transmembrane regulator functions as a chloride (Cl-) channel in multiple organs, including the lungs. More than 1,800 disease-associated mutations have been identified, which can be divided into six classes. In patients with CF due to class III gating mutations, ivacaftor produces significant improvement in lung function, weight, reduction in sweat chloride level, and pulmonary exacerbations by enhancing the probability of chloride channel opening (gating). Although the benefit of ivacaftor in CF due to gating mutations is established, its potential role in patients with CF due to class IV conductance mutations is emerging. We report 6 months' prospective stability of lung function, improved BMI, reduced pulmonary exacerbations, and reduction in sweat chloride level in a patient with severe CF and the class IV R117H mutation. High-resolution CT scan also improved, thus highlighting the potential usefulness of ivacaftor in patients with severe CF due to class IV mutations.

Membrane potential depolarization causes alterations in neuron arrangement and connectivity in cocultures.

BackgroundThe disruption of neuron arrangement is associated with several pathologies. In contrast to action potentials, the role of resting potential (Vmem) in regulating connectivity remains unknown.MethodsNeuron assemblies were quantified when their Vmem was depolarized using ivermectin (Ivm), a drug that opens chloride channels, for 24 h in cocultures with astrocytes. Cell aggregation was analyzed using automated cluster analysis methods. Neural connectivity was quantified based on the identification of isolated somas in phase-contrast images using image processing. Vmem was measured using voltage-sensitive dyes and whole-cell patch clamping. Immunocytochemistry and Western blotting were used to detect changes in the distribution and production of the proteins.ResultsData show that Vmem regulates cortical tissue shape and connectivity. Automated cluster analysis methods revealed that the degree of neural aggregation was significantly increased (0.26 clustering factor vs. 0.21 in controls, P ≤ 0.01). The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm. Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures. Glia cell densities increased (33.3%, P ≤ 0.05) under depolarizing conditions.ConclusionVmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

Is GABA neurotransmission enhanced in auditory thalamus relative to inferior colliculus?

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central auditory system. Sensory thalamic structures show high levels of non-desensitizing extrasynaptic GABAA receptors (GABAARs) and a reduction in the redundancy of coded information. The present study compared the inhibitory potency of GABA acting at GABAARs between the inferior colliculus (IC) and the medial geniculate body (MGB) using quantitative in vivo, in vitro, and ex vivo experimental approaches. In vivo single unit studies compared the ability of half maximal inhibitory concentrations of GABA to inhibit sound-evoked temporal responses, and found that GABA was two to three times (P < 0.01) more potent at suppressing MGB single unit responses than IC unit responses. In vitro whole cell patch-clamp slice recordings were used to demonstrate that gaboxadol, a δ-subunit selective GABAAR agonist, was significantly more potent at evoking tonic inhibitory currents from MGB neurons than IC neurons (P < 0.01). These electrophysiological findings were supported by an in vitro receptor binding assay which used the picrotoxin analog [(3)H]TBOB to assess binding in the GABAAR chloride channel. MGB GABAARs had significantly greater total open chloride channel capacity relative to GABAARs in IC (P < 0.05) as shown by increased total [(3)H]TBOB binding. Finally, a comparative ex vivo measurement compared endogenous GABA levels and suggested a trend towards higher GABA concentrations in MGB than in IC. Collectively, these studies suggest that, per unit GABA, high affinity extrasynaptic and synaptic GABAARs confer a significant inhibitory GABAAR advantage to MGB neurons relative to IC neurons. This increased GABA sensitivity likely underpins the vital filtering role of auditory thalamus.

Opening of Chloride Channels by 1α,25-Dihydroxyvitamin D3 Contributes to Photoprotection against UVR-Induced Thymine Dimers in Keratinocytes

UVR produces vitamin D in skin, which is hydroxylated locally to 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). 1,25(OH)(2)D(3) protects skin cells against UVR-induced DNA damage, including thymine dimers, but the mechanism is unknown. As DNA repair is inhibited by nitric oxide (NO) products but facilitated by p53, we examined whether 1,25(OH)(2)D(3) altered the expression of nitrotyrosine, a product of NO, or p53 after UVR in human keratinocytes. 1,25(OH)(2)D(3) and the nongenomic agonist 1α,25-dihydroxylumisterol(3) reduced nitrotyrosine 16 hours after UVR, detected by a sensitive whole-cell ELISA. p53 was enhanced after UVR, and this was further augmented in the presence of 1,25(OH)(2)D(3). DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a chloride channel blocker previously shown to prevent 1,25(OH)(2)D(3)-induced chloride currents in osteoblasts, had no effect on thymine dimers on its own but prevented the 1,25(OH)(2)D(3)-induced protection against thymine dimers. Independent treatment with DIDS, at concentrations that had no effect on thymine dimers, blocked UVR-induced upregulation of p53. In contrast, reduction of nitrotyrosine remained in keratinocytes treated with 1,25(OH)(2)D(3) and DIDS at concentrations shown to block decreases in post-UVR thymine dimers. These results suggest that 1,25(OH)(2)D(3)-induced chloride currents help protect from UVR-induced thymine dimers, but further increases in p53 or reductions of nitrotyrosine by 1,25(OH)(2)D(3) are unlikely to contribute substantially to this protection.

Chloride currents in cones modify feedback from horizontal cells to cones in goldfish retina

In neuronal systems, excitation and inhibition must be well balanced to ensure reliable information transfer. The cone/horizontal cell (HC) interaction in the retina is an example of this. Because natural scenes encompass an enormous intensity range both in temporal and spatial domains, the balance between excitation and inhibition in the outer retina needs to be adaptable. How this is achieved is unknown. Using electrophysiological techniques in the isolated retina of the goldfish, it was found that opening Ca(2+)-dependent Cl(-) channels in recorded cones reduced the size of feedback responses measured in both cones and HCs. Furthermore, we show that cones express Cl(-) channels that are gated by GABA released from HCs. Similar to activation of I(Cl(Ca)), opening of these GABA-gated Cl(-) channels reduced the size of light-induced feedback responses both in cones and HCs. Conversely, application of picrotoxin, a blocker of GABA(A) and GABA(C) receptors, had the opposite effect. In addition, reducing GABA release from HCs by blocking GABA transporters also led to an increase in the size of feedback. Because the independent manipulation of Ca(2+)-dependent Cl(-) currents in individual cones yielded results comparable to bath-applied GABA, it was concluded that activation of either Cl(-) current by itself is sufficient to reduce the size of HC feedback. However, additional effects of GABA on outer retinal processing cannot be excluded. These results can be accounted for by an ephaptic feedback model in which a cone Cl(-) current shunts the current flow in the synaptic cleft. The Ca(2+)-dependent Cl(-) current might be essential to set the initial balance between the feedforward and the feedback signals active in the cone HC synapse. It prevents that strong feedback from HCs to cones flood the cone with Ca(2)(+). Modulation of the feedback strength by GABA might play a role during light/dark adaptation, adjusting the amount of negative feedback to the signal to noise ratio of the cone output.

Role of the Juxtamembrane Region of Cytoplasmic Loop 3 in the Gating and Conductance of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl– conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl– permeation pathway to exert any influence on permeating Cl– ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl– ions use other routes to access the transmembrane pore.

Functional Modifications of Acid-Sensing Ion Channels by Ligand-Gated Chloride Channels

Together, acid-sensing ion channels (ASICs) and epithelial sodium channels (ENaC) constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that ASICs were reversibly inhibited by activation of GABAA receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABAA receptor-mediated currents. Moreover, activation of the GABAA receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABAA receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABAA receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABAA receptors, also modified ASICs in spinal neurons. We conclude that GABAA receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.