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Chloride Channels (chloride + channel)
Selected AbstractsN-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel,HUMAN MUTATION, Issue 5 2008G.G. Gené Abstract Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure,function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology. Hum Mutat 29(5), 738,749, 2008. © 2008 Wiley-Liss, Inc. [source] Role of a serine residue (S278) in the pore-facing region of the housefly l -glutamate-gated chloride channel in determining sensitivity to noncompetitive antagonistsINSECT MOLECULAR BIOLOGY, Issue 4 2008K. Hirata Abstract ,-Hexachlorocyclohexane (,-HCH), fipronil and picrotoxinin are noncompetitive antagonists (NCAs) of l -glutamate-gated chloride channels (GluCls), yet their potencies are weaker than those on ,-aminobutyric acid receptors (GABARs). The A302S mutation of Drosophila RDL (resistant to dieldrin) GABAR confers NCA resistance, and housefly GluCls (MdGluCls) possess S278 as the residue corresponding to the A302. Thus, the effects of S278A mutation on the NCA actions on MdGluCls were investigated. The S278A mutation resulted in enhanced blocking by NCAs of the MdGluCl response to 30 µM l -glutamate. However, such actions of ,-HCH and picrotoxinin, but not of fipronil, on the S278A mutant were reduced with 200 µM l -glutamate. Further increases in the l -glutamate concentration led to potentiation by NCAs of the mutant response to l -glutamate. [source] Severe Malignant Osteopetrosis Caused by a GL Gene Mutation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2004Paola Quarello Abstract Infantile malignant autosomal recessive osteopetrosis is a genetically heterogeneous disease caused by the inability of OCLs to resorb and remodel bone, resulting in generalized osteosclerosis and obliteration of marrow spaces and cranial foramina. The classical clinical features are pathological fractures, visual impairment, and bone marrow failure. Two human genes have been described as the cause of this form of osteopetrosis: the T-cell immune-regulator-1 (TCIRG1) gene, which is mutated in >50% of the patients, and the chloride channel 7 (ClCN7) gene, which accounts for ,10% of cases. We report the clinical, radiographic, and histopathologic findings of the first human osteopetrosis case caused by a mutation in the grey-lethal (GL) gene. The patient, a 9-day-old male infant, presented with a very severe osteopetrotic phenotype including substantial hepatosplenomegaly since birth, cytopenia, and progressive major liver failure. Skeletal radiographs revealed a generalized increase in bone density with loss of corticomedullary differentiation. Histopathologic bone examination showed the typical osteopetrotic changes, with absence of resorptive activity, and osteoclasts, slightly decreased in number, with evident morphological alterations. [source] Involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in the pathogenesis of hydrosalpinx induced by Chlamydia trachomatis infectionJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 6 2008Louis Chukwuemeka Ajonuma Abstract Background:, Genital Chlamydia (C) trachomatis infection has been recognized as the single most common cause of pelvic inflammatory disease leading to severe tubal damage, ectopic pregnancy, infertility and hydrosalpinx. However, the mechanism underlying the formation of hydrosalpinx induced by C. trachomatis infection remains largely unknown. We performed this study to determine the involvement of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel that regulates epithelial electrolyte and fluid secretion, in hydrosalpinx fluid formation. Methods:, Western blot analysis was used to determine CFTR expression in the hydrosalpinges that were seen on the ultrasound scans of infertile assisted reproduction treatment patients. Correlation with C. trachomatis infection was done by testing patients' sera for C. trachomatis immunoglobulin G antibody titer using a Capita enzyme-linked immunosorbent assay based kit. CFTR involvement was further verified in a rat C. trachomatis infection model and confirmed using CFTR mutant (CFTRtm1Unc) mice. Results:, Here we report on the up-regulated expression of CFTR in the hydrosalpinx tissues of infertile patients with detectable serum levels of C. trachomatis antibody (immunoglobulin G). In a rat model, increased CFTR expression and fluid accumulation could be observed in the uterine horns infected with C. trachomatis elementary bodies, which was reversed by antibiotics treatment. In C. trachomatis,infected CFTRtm1Unc mice, however, no detectable fluid accumulation was observed. Conclusion:, These findings suggest the involvement of CFTR in the pathogenesis of hydrosalpinx fluid formation and may provide grounds for a better treatment strategy to improve assisted reproduction treatment outcome in infertile patients with hydrosalpinx. [source] Photodegradation study of a new activator of the cystic fibrosis chloride channel, the 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07)JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2002Jean-Christophe Olivier Abstract The photodegradation of 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07), a new activator of the transmembrane conductance regulator chloride channel, was studied in aqueous solutions exposed to artificial daylight (2300 Lux intensity). Various conditions of pH, concentration, and temperature were used. MPB-07 concentration was determined at regular time intervals by reversed-phase HPLC. MPB-07 stability was also studied at pH 7.4 in the dark. Results showed that in all the conditions tested MPB-07 underwent rapid photodegradation, apparently following first-order kinetics. Rate constants were dependent on the initial MPB-07 concentration, temperature, and pH. At pH 7.4, and for concentrations from 1 to 125 ,M, half-lives ranged from 0.681,±,0.047 to 4.54,±,0.28 h. The Arrhenius plot was linear and activation energy was calculated to be 20.7 kJ,·,mol,1. Analysis by chemical ionization-mass spectrometry showed that the chlorine atom of the MPB-07 molecule might be replaced by an OH group during the photodegradation process. In the dark, MPB-07 in solutions at pH 7.4 was found to be stable over a 6-week period. In conclusion, MPB-07 is a highly photolabile molecule that should be carefully protected from light when used. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:324,330, 2002 [source] First cloning and functional characterization of a melatonin receptor in fish brain: a novel one?JOURNAL OF PINEAL RESEARCH, Issue 2 2002Pascaline Gaildrat Melatonin, a neuroendocrine transducer of photoperiod, influences a number of physiological functions and behaviors through specific seven transmembrane domains receptors. We report here the first full-length cloning and functional characterization of a melatonin receptor (P2.6) in a fish, the pike (Teleost). P2.6 encodes a protein that is ,80% identical to melatonin receptors previously isolated partially in non-mammals and classified as members of the Mel1b subtype; but, it shares only 61% identity with the full-length human Mel1b melatonin receptor (hMT2). Expression of P2.6 results in ligand binding characteristics similar to that described for endogenous melatonin receptors. Selective antagonists of the hMT2 (4-phenyl-2-propionamidotetraline and luzindole) were poor competitors of 2-[125I]iodomelatonin binding to the recombinant receptor. In Chinese hamster ovary cells expressing both the cystic fibrosis transmembrane conductance regulator chloride channel and P2.6 receptor, melatonin counteracted the forskolin induced activation of the channel. The results are best explained by a selective inhibition of the adenylyl cyclase. By reverse transcription-polymerase chain reaction, P2.6 mRNA appeared expressed in the optic tectum and, to lesser extent, in the retina and pituitary. In conclusion, these results, together with those of a phylogenetic analysis, suggest that P2.6 might belong to a distinct subtype group within the vertebrate melatonin receptor family. [source] Identification of the genes encoding for putative gamma aminobutyric acid (GABA) and glutamate-gated chloride channel (GluCl) alpha receptor subunits in sea lice (Lepeophtheirus salmonis)JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2007N. D. TRIBBLE First page of article [source] Neurological aspects of osteopetrosisNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 2 2003C. G. Steward The osteopetroses are caused by reduced activity of osteoclasts which results in defective remodelling of bone and increased bone density. They range from a devastating neurometabolic disease, through severe malignant infantile osteopetrosis (OP) to two more benign conditions principally affecting adults [autosomal dominant OP (ADO I and II)]. In many patients the disease is caused by defects in either the proton pump [the a3 subunit of vacuolar-type H(+)-ATPase, encoded by the gene variously termed ATP6i or TCIRG1] or the ClC-7 chloride channel (ClCN7 gene). These pumps are responsible for acidifying the bone surface beneath the osteoclast. Although generally thought of as bone diseases, the most serious consequences of the osteopetroses are seen in the nervous system. Cranial nerves, blood vessels and the spinal cord are compressed by either gradual occlusion or lack of growth of skull foramina. Most patients with OP have some degree of optic atrophy and many children with severe forms of autosomal recessive OP are rendered blind; optic decompression is frequently attempted to prevent the latter. Auditory, facial and trigeminal nerves may also be affected, and hydrocephalus can develop. Stenosis of both arterial supply (internal carotid and vertebral arteries) and venous drainage may occur. The least understood form of the disease is neuronopathic OP [OP and infantile neuroaxonal dystrophy, MIM (Mendelian inheritance in man) 600329] which causes rapid neurodegeneration and death within the first year. Although characterized by the finding of widespread axonal spheroids and accumulation of ceroid lipofuscin, the biochemical basis of this disease remains unknown. The neurological complications of this disease and other variants are presented in the context of the latest classification of the disease. [source] CFTR: More than just a chloride channelPEDIATRIC PULMONOLOGY, Issue 4 2005Anil Mehta MBBS, FRCP (Edin), FRCPCH Abstract This review examines the cystic fibrosis transmembrane conductance regulator (CFTR) protein. After summarizing the ion channels regulated by CFTR, the review focuses on the functions of CFTR that do not relate directly to a disease mechanism based on a channelopathy. The key concept is that newly synthesized CFTR has to enter lipid vesicles which bud from the endoplasmic reticulum. This is abnormally low in ,F508 CFTR. Normal wild type vesicular CFTR enters a recycling pool of lipid vesicles which transiently dock with the apical membrane only for CFTR to be retrieved shortly after into a sub-apical recycling compartment. This retrieval is abnormally fast in ,F508 CFTR. The review discusses the relationship between this process and the difficult topic of fat metabolism and then explores the possible links between abnormal fatty acid turnover and inflammatory cascades that are abnormal in cystic fibrosis. Finally the review concentrates on the emerging functions of a protein kinase (AMP-activated kinase) which is bound near the C terminus of the CFTR protein whose functions could intergrate some of the abnormalities in lipid metabolism that result from mislocalization of CFTR in clinical disease. Pediatr Pulmonol. 2005; 39:292,298. © 2004 Wiley-Liss, Inc. [source] Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosisPROTEIN SCIENCE, Issue 10 2010Chi Wang Abstract The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR. [source] Permeant anions contribute to voltage dependence of ClC-2 chloride channel by interacting with the protopore gateTHE JOURNAL OF PHYSIOLOGY, Issue 14 2010Jorge E. Sánchez-Rodríguez It has been shown that the voltage (Vm) dependence of ClC Cl, channels is conferred by interaction of the protopore gate with H+ ions. However, in this paper we present evidence which indicates that permeant Cl, ions contribute to Vm -dependent gating of the broadly distributed ClC-2 Cl, channel. The apparent open probability (PA) of ClC-2 was enhanced either by changing the [Cl,]i from 10 to 200 mm or by keeping the [Cl,]i low (10 mm) and then raising [Cl,]o from 10 to 140 mm. Additionally, these changes in [Cl,] slowed down channel closing at positive Vm suggesting that high [Cl,] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the PA(Vm) curves were nearly identical with Cl, or Br, but shifted to negative voltages in the presence of SCN, ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN,/Cl, mixture in agreement with the idea that pore occupancy by different permeant anions modifies the Vm dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl, ions dwell in the central binding site. In contrast, when Cl, ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl, -dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl,]o enhances channel opening when the [Cl,]i is in the physiological range. [source] Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protonsTHE JOURNAL OF PHYSIOLOGY, Issue 7 2009María Isabel Niemeyer The ClC transport protein family comprises both Cl, ion channel and H+/Cl, and H+/NO3, exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl, passage and in addition serves as a staging post for H+ exchange. This same conserved glutamate acts as a gate to regulate Cl, flow in ClC channels. The activity of ClC-2, a genuine Cl, channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than ,7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl, acts as a voltage-independent modulator, as though regulating the pKa of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl, efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts. [source] Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channelsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000Chantal Maertens It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15,21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, ICl,swell, was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16,21 significantly inhibited ICl,swell (n=4,5). Ca2+ -activated Cl, currents, ICl,Ca, activated by loading T84 cells via the patch pipette with 1 ,M free Ca2+, were not inhibited by any of the tested fractions (15,21), (n=2,5). Chlorotoxin (625 nM) did neither effect ICl,swell nor ICl,Ca (n=4,5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 ,M chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca2+ -activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin. British Journal of Pharmacology (2000) 129, 791,801; doi:10.1038/sj.bjp.0703102 [source] Parasitoid wasp sting: A cocktail of GABA, taurine, and ,-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach hostDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2006Eugene L. Moore Abstract The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists ,-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host. © 2006 Wiley Periodicals, Inc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Developmental shift in bidirectional functions of taurine-sensitive chloride channels during cortical circuit formation in postnatal mouse brainDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2004Mika Yoshida Abstract Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in the developing mammalian cerebral cortex, however, few studies have reported its neurobiological functions during development. In this study, by means of whole-cell patch-clamp recordings, we examined the effects of taurine on chloride channel receptors in neocortical neurons from early to late postnatal stages, which cover a critical period in cortical circuit formation. We show here that taurine activates chloride channels in cortical neurons throughout the postnatal stages examined (from postnatal day 2 to day 36). The physiological effects of taurine changed from excitatory to inhibitory due to variations in the intracellular Cl, concentration during development. An antagonist blocking analysis also demonstrated a developmental shift in the receptor target of taurine, from glycine receptors to GABAA receptors. Taken together, these results may reflect genetically programmed, bidirectional functions of taurine. At the early developmental stage, taurine acting on glycine receptors would serve to promote cortical circuit formation. As cortical circuit has to be regulated in the later stages, taurine would serve as a safeguard against hyperexcitable circuit. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 166,175, 2004 [source] Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2001Dominique Chesnoy-Marchais Abstract Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive l -type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 µm, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10,50 µm), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels. [source] Synaptic stimulation of nicotinic receptors in rat sympathetic ganglia is followed by slow activation of postsynaptic potassium or chloride conductancesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000Oscar Sacchi Abstract Two slow currents have been described in rat sympathetic neurons during and after tetanization of the whole preganglionic input. Both effects are mediated by nicotinic receptors activated by native acetylcholine (ACh). A first current, indicated as IAHPsyn, is calcium dependent and voltage independent, and is consistent with an IAHP -type potassium current sustained by calcium ions accompanying the nicotinic synaptic current. The conductance activated by a standard synaptic train was ,,3.6 nS per neuron; it was detected in isolation in 14 out of a 52-neuron sample. A novel current, IADPsyn, was described in 42/52 of the sample as a post-tetanic inward current, which increased in amplitude with increasing membrane potential negativity and exhibited a null-point close to the holding potential and the cell momentary chloride equilibrium potential. IADPsyn developed during synaptic stimulation and decayed thereafter according to a single exponential (mean ,,= 148.5 ms) in 18 neurons or according to a two-exponential time course (, = 51.8 and 364.9 ms, respectively) in 19 different neurons. The mean peak conductance activated was ,,20 nS per neuron. IADPsyn was calcium independent, it was affected by internal and external chloride concentration, but was insensitive to specific blockers (anthracene-9-carboxylic acid, 9AC) of the chloride channels open in the resting neuron. It is suggested that gADPsyn represents a specific chloride conductance activatable by intense nicotinic stimulation; in some neurons it is even associated with single excitatory postsynaptic potentials (EPSCs). Both IAHP and IADPsyn are apparently devoted to reduce neuronal excitability during and after intense synaptic stimulation. [source] Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitroEXPERIMENTAL DERMATOLOGY, Issue 4 2007Mónika Gönczi Abstract:, Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of ,27 ± 4 to ,51 ± 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases. [source] Mrp2 modulates the activity of chloride channels in isolated hepatocytesHEPATOLOGY, Issue 1 2002Xinhua Li Adenosine triphosphate binding cassette family transport proteins are important organic ion transporters in hepatocytes but these molecules may also exhibit other functions. In the present study we have measured the effects of substrates of the canalicular organic ion transporter multidrug resistance associated protein 2 (Mrp2) on chloride channel activation and cell volume regulation. We found that substrates such as leukotriene D4, 17-,-estradiol glucuronide, and the leukotriene inhibitor MK-571 accelerated the activation of chloride channels by cell swelling and activated chloride channels in cytokine-pretreated hepatocytes. Two conjugated estrogens that are not Mrp2 substrates did not produce this effect. Hepatocytes derived from a strain of transport-deficient rats (TR,), which lack Mrp2 expression, showed none of these substrate effects. Coincident with their ability to activate channels, the Mrp2 substrates increased the rate of volume regulatory decrease by approximately 50% (P < .01), confirming that enhanced channel activation under this condition stimulated volume regulation. In TR-hepatocytes the Mrp2 substrate had no effect on volume regulation. In conclusion, Mrp2 plays a role in regulation of chloride channel function by reducing the lag time necessary for channel activation and consequently accelerating the process of cell volume regulation. Substrates of Mrp2 affect the ability of the protein to interact with chloride channels. These findings represent an alternative function of Mrp2 in hepatocytes. [source] Role of a serine residue (S278) in the pore-facing region of the housefly l -glutamate-gated chloride channel in determining sensitivity to noncompetitive antagonistsINSECT MOLECULAR BIOLOGY, Issue 4 2008K. Hirata Abstract ,-Hexachlorocyclohexane (,-HCH), fipronil and picrotoxinin are noncompetitive antagonists (NCAs) of l -glutamate-gated chloride channels (GluCls), yet their potencies are weaker than those on ,-aminobutyric acid receptors (GABARs). The A302S mutation of Drosophila RDL (resistant to dieldrin) GABAR confers NCA resistance, and housefly GluCls (MdGluCls) possess S278 as the residue corresponding to the A302. Thus, the effects of S278A mutation on the NCA actions on MdGluCls were investigated. The S278A mutation resulted in enhanced blocking by NCAs of the MdGluCl response to 30 µM l -glutamate. However, such actions of ,-HCH and picrotoxinin, but not of fipronil, on the S278A mutant were reduced with 200 µM l -glutamate. Further increases in the l -glutamate concentration led to potentiation by NCAs of the mutant response to l -glutamate. [source] Electrophysiological characterization of electrolyte and nutrient transport across the small intestine in horsesJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 3 2009A. Cehak Summary The aim of this study was to characterize the transport mechanisms of electrolytes and nutrients across the jejunum of nine healthy horses electrophysiologically. The stripped mucosa was mounted in Ussing chambers and tissue conductances (Gt) and short circuit currents (Isc) were continuously monitored. After blocking the sodium and potassium channels with amiloride, tetraethylammonium chloride (TEA) and barium, chloride secretion was stimulated by carbachol and forskolin. Subsequently, chloride channels were inhibited by 4,4,-diisothiocyanato-stilbene-2,2,-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, CFTRinh -172, N -(2-naphtalenyl)-(3.5-dibromo-2.4-dihydroxyphenyl)methylene glycine hydrazide (GlyH-101) and glibenclamide and their dose,response effect was investigated. The response to glucose, l -alanine and glycyl- l -glutamine was determined at two different mucosal pH values (pH 7.4 and 5.4 respectively). Mean basal Isc was ,0.47 ± 0.31 ,Eq/cm2h and mean Gt was 22.17 ± 1.78 mS/cm2. Amiloride and TEA did not alter the baseline Isc. Barium, carbachol and forskolin significantly increased Isc. Irrespective of the dose, none of the chloride inhibitors changed Isc. All nutrients induced a significant increase in Isc with the increase being significantly higher at pH 7.4 than at pH 5.4. In conclusion, there is evidence that chloride secretion in horses may be different from respective transport mechanisms in other species. The glucose absorption is suggestive of a sodium-dependent glucose cotransporter 1. However, a decrease in luminal pH did not stimulate current response to peptides as shown for other mammals. [source] Glial cell-derived glutamate mediates autocrine cell volume regulation in the retina: activation by VEGFJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Antje Wurm Abstract Astroglial cells are a source for gliotransmitters such as glutamate and ATP. We demonstrate here that gliotransmitters have autocrine functions in the regulation of cellular volume. Hypoosmotic stress in the presence of inflammatory mediators or oxidative stress, and during blockade or down-regulation of potassium channels, induces swelling of retinal glial cells. Vascular endothelial growth factor inhibits the osmotic swelling of glial cells in retinal slices or isolated cells. This effect was mediated by a kinase domain region/flk-1 receptor-evoked calcium dependent release of glutamate from glial cells, and subsequent stimulation of glial group I/II metabotropic glutamate receptors. Activation of kinase domain region/flk-1 or glutamate receptors evoked an autocrine swelling-inhibitory purinergic signaling cascade that was calcium-independent. This cascade involved the release of ATP and adenosine, and the activation of purinergic P2Y1 and adenosine A1 receptors, resulting in the opening of potassium and chloride channels and inhibition of cellular swelling. The glutamatergic-purinergic regulation of the glial cell volume may be functionally important in the homeostasis of the extracellular space volume during intense neuronal activation which is associated with a swelling of neuronal cell structures in the retina. However, glial cell-derived glutamate may also contribute to the swelling of activated neurons since metabolic poisoning of glial cells by iodoacetate inhibits the neuronal cell swelling mediated by activation of ionotropic glutamate receptors. [source] Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptorsTHE JOURNAL OF PHYSIOLOGY, Issue 23 2009Andrew A. VossArticle first published online: 30 NOV 200 ATP is released from skeletal muscle during exercise, a discovery dating back to 1969. Surprisingly, few studies have examined the effects of extracellular ATP on mature mammalian skeletal muscle. This electrophysiological study examined the effects of extracellular ATP on fully innervated rat levator auris longus using two intracellular microelectrodes. The effects of ATP were determined by measuring the relative changes of miniature endplate potentials (mEPPs) and voltage responses to step current pulses in individual muscle fibres. Exposure to ATP (20 ,m) prolonged the mEPP falling phase by 31 ± 7.5% (values ±s.d., n= 3 fibres). Concurrently, the input resistance increased by 31 ± 2.0% and the time course of the voltage responses increased by 59 ± 3.0%. Analogous effects were observed using 2 and 5 ,mATP, and on regions distal from the neuromuscular junction, indicating that physiologically relevant levels of ATP enhanced electrical signalling over the entire muscle fibre. The effects of extracellular ATP were blocked by 200 ,manthracene-9-carboxylic acid, a chloride channel inhibitor, and reduced concentrations of extracellular chloride, indicating that ATP inhibited chloride channels. A high affinity agonist for P2Y receptors, 2-methylthioadenosine-5,- O -diphosphate (2MeSADP), induced similar effects to ATP with an EC50 of 160 ± 30 nm. The effects of 250 nm2MeSADP were blocked by 500 nmMRS2179, a specific P2Y1 receptor inhibitor, suggesting that ATP acts on P2Y1 receptors to inhibit chloride channels. The inhibition of chloride channels by extracellular ATP has implications for muscle excitability and fatigue, and the pathophysiology of myotonias. [source] Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channelsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000Chantal Maertens It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15,21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, ICl,swell, was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16,21 significantly inhibited ICl,swell (n=4,5). Ca2+ -activated Cl, currents, ICl,Ca, activated by loading T84 cells via the patch pipette with 1 ,M free Ca2+, were not inhibited by any of the tested fractions (15,21), (n=2,5). Chlorotoxin (625 nM) did neither effect ICl,swell nor ICl,Ca (n=4,5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 ,M chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca2+ -activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin. British Journal of Pharmacology (2000) 129, 791,801; doi:10.1038/sj.bjp.0703102 [source] Molecular Characteristics And Functional Diversity Of Clca Family MembersCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 11 2000Bendicht U Pauli SUMMARY 1. In the present brief review, we describe some of the molecular and functional characteristics of a novel mammalian family of putative Ca2+ -activated chloride channels (CLCA). 2. So far, two bovine (bCLC1; bCLCA2 (Lu-ECAM-1)), three mouse (mCLCA1; mCLCA2; mCLCA3) and four human (hCLCA1; hCLCA2; hCLCA3; hCLCA4) CLCA family members have been cloned. Each CLCA exhibits a distinct, often overlapping, tissue expression pattern. 3. With the exception of the truncated secreted hCLCA3, all CLCA proteins are synthesized as an approximately 125 kDa precursor transmembrane glycoprotein that is rapidly cleaved into 90 and 35 kDa subunits. 4. The CLCA proteins expressed on the luminal surface of lung vascular endothelia (bCLCA2; mCLCA1; hCLCA2) serve as adhesion molecules for lung metastatic cancer cells, mediating vascular arrest and lung colonization. 5. Expression of hCLCA2 in normal mammary epithelium is consistently lost in human breast cancer and in all tumorigenic breast cancer cell lines. Re-expression of hCLCA2 in human breast cancer cells abrogates invasiveness of Matrigel (BD Biosciences-Labware, Bedford, MA, USA) in vitro and tumorigenicity in nude mice, implying that hCLCA2 acts as a tumour suppressor in breast cancer. [source] | ||||||||||||||||