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Anion Channels (anion + channel)
Selected AbstractsFunctional Characterisation of the Volume-Sensitive Anion Channel in Rat Pancreatic ,-CellsEXPERIMENTAL PHYSIOLOGY, Issue 2 2001L. Best The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic ,-cells. The channel showed high permeability (P) relative to Cl, to extracellular monovalent organic anions (PSCN/PCll= 1.73, Pacetate/PCll= 0.39, Plactate/PCll= 0.38, Pacetoacetate/PCll= 0.32, Pglutamate/PCll= 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll= 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 ,M) and 4-OH tamoxifen (10 ,M). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3,,5,-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic ,-cells can be activated by protein kinase A. Furthermore, the ,-cell channel does not appear to be subject to regulation via protein kinase C. [source] A role for the volume regulated anion channel in volume regulation in the murine CNS cell line, CADACTA PHYSIOLOGICA, Issue 2 2010V. L. Harvey Abstract Aim:, The role of the volume regulated anion channel (VRAC) in a model CNS neuronal cell line, CAD, was investigated. Methods:, Changes in cell volume following hypotonic challenges were measured using a video-imaging technique. The effect of the Cl, channel antagonists tamoxifen (10 ,m) and 4,4,-diisothiocyanatostilbene-2,2,-disulphonic acid (DIDS; 100 ,m) on regulatory volume decrease (RVD) were measured. The whole-cell voltage-clamp technique was used to characterize IClswell, the current underlying the VRAC. Results:, Using the video-imaging technique, CAD cells were found to swell and subsequently exhibit RVD when subjected to a sustained hypotonic challenge from 300 mOsmol kg,1 H2O to 210 mOsmol kg,1 H2O. In the presence of tamoxifen (10 ,m) or DIDS (100 ,m) RVD was abolished, suggesting a role for the VRAC. A hypotonic solution (230 mOsmol kg,1 H2O) evoked IClswell, an outwardly rectifying current displaying time-independent activation, which reversed upon return to isotonic conditions. The reversal potential (Erev) for IClswell was ,14.7 ± 1.4 mV, similar to the theoretical Erev for a selective Cl, conductance. IClswell was inhibited in the presence of DIDS (100 ,m) and tamoxifen (10 ,m), the DIDS inhibition being voltage dependent. Conclusions:, Osmotic swelling elicits an outwardly rectifying Cl, conductance in CAD cells. The IClswell observed in these cells is similar to that observed in other cells, and is likely to provide a pathway for the loss of Cl, which leads to water loss and RVD. As ischaemia, brain trauma, hypoxia and other brain pathologies can cause cell swelling, CAD cells represent a model cell line for the study of neuronal cell volume regulation. [source] Functional Characterisation of the Volume-Sensitive Anion Channel in Rat Pancreatic ,-CellsEXPERIMENTAL PHYSIOLOGY, Issue 2 2001L. Best The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic ,-cells. The channel showed high permeability (P) relative to Cl, to extracellular monovalent organic anions (PSCN/PCll= 1.73, Pacetate/PCll= 0.39, Plactate/PCll= 0.38, Pacetoacetate/PCll= 0.32, Pglutamate/PCll= 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll= 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 ,M) and 4-OH tamoxifen (10 ,M). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3,,5,-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic ,-cells can be activated by protein kinase A. Furthermore, the ,-cell channel does not appear to be subject to regulation via protein kinase C. [source] Inflammatory bowel disease-associated gene expression in intestinal epithelial cells by differential cDNA screening and mRNA displayINFLAMMATORY BOWEL DISEASES, Issue 5 2003Kouhei Fukushima M.D., Ph.D. Abstract Intestinal epithelial cells are actively involved in the pathogenesis of inflammatory bowel disease resulting in an altered functional phenotype. The modulation of epithelial gene expression may occur as a consequence of proliferative, metabolic, immune, inflammatory, or genetic abnormalities. Differential screening of epithelial-cell-derived cDNA libraries (from control, ulcerative colitis, and Crohn's disease epithelial cells) and differential display of mRNA were used for investigation of disease-associated gene expression and modulation. Intestinal epithelial gene expression was successfully analyzed by both approaches. Using differential screening with clones encoding mitochondrial genes, quantitative overexpression was observed in both ulcerative colitis and Crohn's disease, while a unique expression of small RNA was noticed in Crohn's disease cells using Alu-homologous clones. Differential display demonstrated that several genes were differentially displayed among control, ulcerative colitis, and Crohn's disease epithelial cells. This was confirmed by immunohistochemical staining of pleckstrin, desmoglein 2 and voltage-dependent anion channel in control and inflammatory bowel disease mucosal samples. In summary, several inflammatory bowel disease-related associations were found. Since both differential screening and display have advantages and limitations, the combination of both techniques can generate complementary information, facilitate search for novel genes, and potentially identify genes uniquely associated with inflammatory bowel disease. [source] Two conventional protein kinase C isoforms, , and ,I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Alena Rudkouskaya Abstract Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor-dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re-evaluate the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (siRNA) and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 ,M ATP synergistically increased the release of excitatory amino acids, measured with a non-metabolized analog of l -glutamate, d -[3H]aspartate. Both Go6976, the selective inhibitor of Ca2+ -sensitive PKC,, ,I/II, and ,, and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKC, and ,I/II, reduced the effects of ATP on d -[3H]aspartate release by ,45,55%. Similar results were obtained with a mixture of siRNAs targeting rat PKC, and ,I. Surprisingly, down-regulation of individual , and ,I PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume-sensitive excitatory amino acid release via cooperative activation of PKC, and ,I. [source] Proteomic changes in the crucian carp brain during exposure to anoxiaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2009Richard W. Smith Dr. Abstract During exposure to anoxia, the crucian carp brain is able to maintain normal overall protein synthesis rates. However, it is not known if there are alterations in the synthesis or expression of specific proteins. This investigation addresses this issue by comparing the normoxic and anoxic brain proteome. Nine proteins were found to be reduced by anoxia. Reductions in the glycolytic pathway proteins creatine kinase, fructose biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and lactate dehydrogenase reflect the reduced production and requirement for adenosine tri-phosphate during anoxia. In terms of neural protection, voltage-dependent anion channel, a protein associated with neuronal apoptosis, was reduced, along with gefiltin, a protein associated with the subsequent need for neuronal repair. Additionally the expression of proteins associated with neural degeneration and impaired cognitive function also declined; dihydropyrimidinase-like protein-3 and vesicle amine transport protein-1. One protein was found to be increased by anoxia; pre-proependymin, the precursor to ependymin. Ependymin fulfils multiple roles in neural plasticity, memory formation and learning, neuron growth and regeneration, and is able to reverse the possibility of apoptosis, thus further protecting the anoxic brain. [source] CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005G. Nagel The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR,ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain. [source] Identification of Evolutionary Conserved Mouse Sperm Surface Antigens by Human Antisperm Antibodies (ASA) from Infertile PatientsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2006Agnieszka Paradowska Problem The presence of antisperm antibodies (ASA) in semen may impair sperm function leading to immunological infertility. The aim of the study was to identify the evolutionary conserved antigens on mouse sperm surface that react with human ASA in order to study the mechanism of autoimmune infertility. Methods of study The binding of human ASA to mouse sperm was investigated by means of indirect immunofluorescence. 2D-electrophoresis was applied to separate the biotin-labelled mouse membrane proteins using isoelectric focusing followed by polyacrylamide gel electrophoresis. Cognate antigens of ASA from seminal plasma of infertile patients were analysed by Western blotting. Performing avidin-blots it was detected which of the proteins recognized were sperm surface proteins. The spots of interest were analysed by means of mass spectrometry. Results ASA bound most frequently (36%) to the post-acrosomal region and to the midpiece of mouse spermatozoa. About 30% of ASA recognized apo lactate dehydrogenase (LDHC4) as a cognate antigen, 30% voltage-dependent anion channel (VDAC2). ASA of 20% bound to outer dense fibre protein and 20% of samples recognized glutathione S-transferase mu5. Conclusions Human ASA bound to specific cognate antigens of mouse spermatozoa, offering the possibility to study their functional relevance in the mouse model. [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] The Vibrio cholerae haemolysin anion channel is required for cell vacuolation and deathCELLULAR MICROBIOLOGY, Issue 7 2002Monica Moschioni Summary Several strains of Vibrio cholerae secrete a haemolytic toxin of 63 kDa, termed V. cholerae cytolysin (VCC). This toxin causes extensive vacuolation and death of cells in culture and forms an anion-selective channel in planar lipid bilayers and in cells. Here, we identify inhibitors of the VCC anion channel and show that the formation of the anion channel is necessary for the development of the vacuoles and for the cell death induced by this toxin. Using markers of cell organelles, we show that vacuoles derive from different intracellular compartments and we identify the contribution of late endosomes and of the trans -Golgi network in vacuole biogenesis. [source] Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2004Jennifer E. Slemmer Abstract The homeostasis of intracellular Cl, concentration ([Cl,]i) is critical for neuronal function, including ,-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl,]i using a transgenetically expressed Cl, -sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 µm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl, -free solution, demonstrating that the EYFP signal represented an increase in [Cl,]i. Similar to glutamate, a rise in [Cl,]i was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K+], indicating that an increase in driving force for Cl, suffices to increase [Cl,]i. To elucidate the membrane mechanisms mediating the Cl, influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl,]i was resistant to furosemide, bumetanide and 4,4,-diisothiocyanato-stilbene-2,2,-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl,]i via several pathways involving NFA- and NPPB-sensitive anion channels and GABAA receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl, transporters. The present study highlights the vulnerability of [Cl,]i homeostasis after membrane depolarization in neurons. [source] Postnatal maturation of Na+, K+, 2Cl, cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2002Serge Marty Abstract GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl, through GABAA -gated anion channels. The outward Cl, gradient that provides the driving force for Cl, efflux might be generated and maintained by the Na+, K+, 2Cl, cotransporter (NKCC) that keeps intracellular Cl, concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing. [source] Pharmacological "cross-inhibition" of connexin hemichannels and swelling activated anion channelsGLIA, Issue 3 2009Zu-Cheng Ye Abstract The study of ion channels has relied heavily on the use of pharmacological blocking agents. However, many of these agents have multiple effects, which may compromise interpretation of results when the affected mechanisms/pathways mediate similar functions. Volume regulated anion channels (VRAC) and connexin hemichannels can both mediate the release of glutamate and taurine, although these channels have distinct activation stimuli and hemichannels, but not VRAC, are permeable to Lucifer Yellow (LY). It has been reported that some anion channel blockers may inhibit connexin hemichannels. We further examined the effects of classic gap junction/hemichannel blockers and anion channel blockers on these channels. The typical VRAC blockers, NPPB, IAA-94, and tamoxifen blocked low divalent cation-induced glutamate and taurine release and LY loading, presumed due to hemichannel opening. The blocking action of these compounds on hemichannels was concentration dependent and fell within the same range where the drugs classically block VRACs. Conversely, carbenoxolone (CBX), the most widely used gap junction/hemichannel blocker, was an effective blocker of VRAC-mediated glutamate and taurine release, and blocked these channels at similar concentrations at which it blocked hemichannels. The CBX effect on VRACs was verified using astrocytes from connexin 43 knock out (Cx43 KO) animals. In these cells, the hypotonic induced amino acid flux was retained whereas the low divalent cation solution-induced flux was lost. These results extend our knowledge about "cross-inhibition" of VRACs and gap junctions/hemichannels by certain pharmacological agents. Given the overlap in function of these two types of channels, great care must be exerted in using pharmacological blockers to identify one channel from the other. © 2008 Wiley-Liss, Inc. [source] Chlorotoxin-sensitive Ca2+ -activated Cl, channel in type R2 reactive astrocytes from adult rat brainGLIA, Issue 4 2003Stanislava Dalton Abstract Astrocytes express four types of Cl, or anion channels, but Ca2+ -activated Cl, (ClCa) channels have not been described. We studied Cl, channels in a morphologically distinct subpopulation (, 5% of cells) of small (10,12 ,m, 11.8 ± 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, ,57.1 ± 4.0 mV, polarized to ,72.7 ± 4.5 mV with BAPTA-AM, an intracellular Ca2+ chelator, and depolarized to ,30.7 ± 6.1 mV with thapsigargin, which mobilizes Ca2+ from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl, reversal potential, which was blocked by Cl, channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn2+, by I, (10 mM), and by chlorotoxin (EC50 = 47 nM). With conventional whole-cell clamp, NPPB- and Zn2+ -sensitive currents became larger with increasing [Ca2+]i (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca2+ sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive ClCa channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive ClCa channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte. GLIA 42:325,339, 2003. © 2003 Wiley-Liss, Inc. [source] Two conventional protein kinase C isoforms, , and ,I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Alena Rudkouskaya Abstract Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor-dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re-evaluate the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (siRNA) and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 ,M ATP synergistically increased the release of excitatory amino acids, measured with a non-metabolized analog of l -glutamate, d -[3H]aspartate. Both Go6976, the selective inhibitor of Ca2+ -sensitive PKC,, ,I/II, and ,, and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKC, and ,I/II, reduced the effects of ATP on d -[3H]aspartate release by ,45,55%. Similar results were obtained with a mixture of siRNAs targeting rat PKC, and ,I. Surprisingly, down-regulation of individual , and ,I PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume-sensitive excitatory amino acid release via cooperative activation of PKC, and ,I. [source] Mechanisms of glutamate release elicited in rat cerebrocortical nerve endings by ,pathologically' elevated extraterminal K+ concentrationsJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Luca Raiteri Abstract Extracellular [K+] can increase during some pathological conditions, resulting into excessive glutamate release through multiple mechanisms. We here investigate the overflow of [3H]d -aspartate ([3H] d -ASP) and of endogenous glutamate elicited by increasing [K+] from purified rat cerebrocortical synaptosomes. Depolarization with [K+] , 15 mmol/L provoked [3H] d -ASP and glutamate overflows almost totally dependent on external Ca2+. Consistent with release by exocytosis, the overflow of [3H] d -ASP evoked by 12 mmol/L K+ was sensitive to clostridial toxins. The overflows evoked by 35/50 mmol/L K+ remained external Ca2+ -dependent by more than 50%. The Ca2+ -independent components of the [3H] d -ASP overflows evoked by [K+] > 15 mmol/L were prevented by the glutamate transporter inhibitors dl - threo -beta-benzyloxyaspartate (dl -TBOA) and dihydrokainate. Differently, the overflows of endogenous glutamate provoked by [K+] > 15 mmol/L were insensitive to both inhibitors; the external Ca2+ -independent glutamate overflow caused by 50 mmol/L KCl was prevented by bafilomycin, by chelating intraterminal Ca2+, by blocking the mitochondrial Na+/Ca2+ exchanger and, for a small portion, by blocking anion channels. In contrast to purified synaptosomes, the 50 mmol/L K+ -evoked release of endogenous glutamate or [3H]D-ASP was inhibited by dl -TBOA in crude synaptosomes; moreover, it was external Ca2+ -insensitive and blocked by dl -TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia. [source] Physiological characteristics of selenite uptake by maize roots in response to different pH levelsJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 3 2010Lianhe Zhang Abstract In selenite solutions, H2SeO3, HSeO, and SeO<$>_3^{2-}<$> are in equilibrium in proportions that vary with solution pH. The physiological characteristics of selenite uptake were studied with excised roots of maize (Zea mays L.) seedlings at pH 3.0, 5.0, and 8.0. The results showed that 0.10 mM 2,4-dinitrophenol (DNP), 1.0 mM sodium fluoride (NaF), and a temperature of 4°C inhibited selenite uptake by maize roots by 16%, 20%, and 23% at pH 3.0, by up to 80%, 79%, and 78% at pH 5.0, and by 5%, 9%, and 16% at pH 8.0. Hence, selenite may enter roots at pH 5.0 in an energy-dependent manner, in contrast to pH 3.0 and 8.0. The uptake kinetics for selenite were determined for excised roots of maize, and the curves were linear at pH 3.0 and 8.0, but saturated at pH 5.0, showing that carrier-mediated uptake of selenite occurred at pH 5.0, but not at pH 3.0 or 8.0. Further studies showed that HgCl2 and AgNO3 inhibited selenite uptake separately by 81% and 76% at pH 3.0 and indicated that selenite was absorbed by maize roots through aquaporins at pH 3.0. At pH 8.0, anion-channel inhibitors only inhibited a small fraction of selenite uptake, indicating that the major absorption pathway of SeO<$>_3^{2-}<$> species into roots was not absorbed passively through anion channels, but might involve other processes. According to these results, it is proposed that selenite uptake occurs via different mechanisms depending on its species in solution in response to pH levels. [source] Diverse transport modes by the solute carrier 26 family of anion transportersTHE JOURNAL OF PHYSIOLOGY, Issue 10 2009Ehud Ohana The solute carrier 26 (SLC26) transporters are anion transporters with diverse substrate specificity. Several members are ubiquitous while others show limited tissue distribution. They are expressed in many epithelia and to the extent known, play a central role in anion secretion and absorption. Members of the family are primarily Cl, transporters, although some members transport mainly SO42,, Cl,, HCO3, or I,. A defining feature of the family is their functional diversity. Slc26a1 and Slc26a2 function as specific SO42, transporters while Slc26a4 functions as an electroneutral Cl,/I,/HCO3, exchanger. Slc26a3 and Slc26a6 function as coupled electrogenic Cl,/HCO3, exchangers or as bona fide anion channels. SLC26A7 and SLC26A9 function exclusively as Cl, channels. This short review discusses the functional diversity of the SLC26 transporters. [source] Spontaneously active and InsP3 -activated ion channels in cell nuclei from rat cerebellar Purkinje and granule neuronesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Sergey M. Marchenko Increases in Ca2+ concentration in the nucleus of neurones modulate gene transcription and may be involved in activity-dependent long-term plasticity, apoptosis, and neurotoxicity. Little is currently known about the regulation of Ca2+ in the nuclei of neurones. Investigation of neuronal nuclei is hampered by the cellular heterogeneity of the brain where neurones comprise no more than 10% of the cells. The situation is further complicated by large differences in properties of different neurones. Here we report a method for isolating nuclei from identified central neurones. We employed this technique to study nuclei from rat cerebellar Purkinje and granule neurones. Patch-clamp recording from the nuclear membrane of Purkinje neurones revealed numerous large-conductance channels selective for monovalent cations. The nuclear membrane of Purkinje neurones also contained multiple InsP3 - activated ion channels localized exclusively in the inner nuclear membrane with their receptor loci facing the nucleoplasm. In contrast, the nuclear membrane of granule neurones contained only a small number of mainly anion channels. Nuclear InsP3 receptors (InsP3Rs) were activated by InsP3 with EC50= 0.67 ,m and a Hill coefficient of 2.5. Ca2+ exhibited a biphasic effect on the receptors elevating its activity at low concentrations and inhibiting it at micromolar concentrations. InsP3 in saturating concentrations did not prevent the inhibitory effect of Ca2+, but strongly increased InsP3R activity at resting Ca2+ concentrations. These data are the first evidence for the presence of intranuclear sources of Ca2+ in neurones. Ca2+ release from the nuclear envelope may amplify Ca2+ transients penetrating the nucleus from the cytoplasm or generate Ca2+ transients in the nucleus independently of the cytoplasm. [source] Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+ -associated opening of plasma membrane anion channelsTHE PLANT JOURNAL, Issue 3 2010Elena Jeworutzki Summary The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode-impaled mesophyll cells and root hairs of Col-0 Arabidopsis plants revealed rapid, dose-dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion-selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species-independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2-associated receptor-like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1-dependent, calcium-associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway. [source] Phosphatidylinositol 4,5-bisphosphate is important for stomatal openingTHE PLANT JOURNAL, Issue 5 2007Yuree Lee Summary Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P2, decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P2 modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P2 -binding domain GFP:PLC,1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P2 increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P2 substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P2 targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P2. Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P2 in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P2. The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P2 in irradiated guard cells, inhibiting anion channels to allow full stomatal opening. [source] Unexpected roles for cryptochrome 2 and phototropin revealed by high-resolution analysis of blue light-mediated hypocotyl growth inhibitionTHE PLANT JOURNAL, Issue 5 2001Kevin M. Folta Summary Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic response known as de-etiolation, the transformation of a dark-grown seedling into a pigmented, photoautotrophic organism. In Arabidopsis thaliana, high-resolution studies of hypocotyl growth accomplished by computer-assisted electronic image capture and analysis revealed that inhibition occurs in two genetically independent phases, the first beginning within 30 sec of illumination. The present work demonstrates that phototropin (nph1), the photoreceptor responsible for phototropism, is largely responsible for the initial, rapid inhibition. Signaling from phototropin during the curvature response is dependent upon interaction with NPH3, but the results presented here demonstrate that NPH3 is not necessary for phototropin-dependent growth inhibition. Activation of anion channels, which transiently depolarizes the plasma membrane within seconds of BL, is an early event in the cryptochrome signaling pathway leading to a phase of growth inhibition that replaces the transient phototropin-dependent phase after approximately 30 min of BL. Surprisingly, cry1 and cry2 were found to contribute equally and non-redundantly to anion-channel activation and to growth inhibition between 30 and 120 min of BL. Inspection of the inhibition kinetics displayed by nph1 and nph1cry1 mutants revealed that the cryptochrome phase of inhibition is delayed in seedlings lacking phototropin. This result indicates that BL-activation of phototropin influences cryptochrome signaling leading to growth inhibition. Mutations in the NPQ1 gene, which inhibit BL-induced stomatal opening, do not affect any aspect of the growth inhibition within the first 120 min examined here, and NPQ1 does not affect the activation of anion channels. [source] Dynamics of ionic activities in the apoplast of the sub-stomatal cavity of intact Vicia faba leaves during stomatal closure evoked by ABA and darknessTHE PLANT JOURNAL, Issue 3 2000Hubert H. Felle Summary Stomatal movement is accomplished by changes in the ionic content within guard cells as well as in the cell wall of the surrounding stomatal pore. In this study, the sub-stomatal apoplastic activities of K+, Cl,, Ca2+ and H+ were continuously monitored by inserting ion-selective micro-electrodes through the open stomata of intact Vicia faba leaves. In light-adapted leaves, the mean activities were 2.59 mm (K+), 1.26 mm (Cl,), 64 µm (Ca2+) and 89 µm (H+). Stomatal closure was investigated through exposure to abscisic acid (ABA), sudden darkness or both. Feeding the leaves with ABA through the cut petiole initially resulted in peaks after 9,10 min, in which Ca2+ and H+ activities transiently decreased, and Cl, and K+ activities transiently increased. Thereafter, Ca2+, H+ and Cl, activities completely recovered, while K+ activity approached an elevated level of around 10 mm within 20 min. Similar responses were observed following sudden darkness, with the difference that Cl, and Ca2+ activities recovered more slowly. Addition of ABA to dark-adapted leaves evoked responses of Cl, and Ca2+ similar to those observed in the light. K+ activity, starting from its elevated level, responded to ABA with a transient increase peaking around 16 mm, but then returned to its dark level. During stomatal closure, membrane potential changes in mesophyll cells showed no correlation with the K+ kinetics in the sub-stomatal cavity. We thus conclude that the increase in K+ activity mainly resulted from K+ release by the guard cells, indicating apoplastic compartmentation. Based on the close correlation between Cl, and Ca2+ changes, we suggest that anion channels are activated by a rise in cytosolic free Ca2+, a process which activates depolarization-activated K+ release channels. [source] | ||||||||||||||||||||||||||||