Activation Of Anion Channels Research Articles

SWELL1, a Plasma Membrane Protein, Is an Essential Component of Volume-Regulated Anion Channel

Maintenance of a constant cell volume in response to extracellular or intracellular osmotic changes is critical for cellular homeostasis. Activation of a ubiquitous volume-regulated anion channel (VRAC) plays a key role in this process; however, its molecular identity in vertebrates remains unknown. Here, we used a cell-based fluorescence assay and performed a genome-wide RNAi screen to find components of VRAC. We identified SWELL1 (LRRC8A), a member of a four-transmembrane protein family with unknown function, as essential for hypotonicity-induced iodide influx. SWELL1 is localized to the plasma membrane, and its knockdown dramatically reduces endogenous VRAC currents and regulatory cell volume decrease in various cell types. Furthermore, point mutations in SWELL1 cause a significant change in VRAC anion selectivity, demonstrating that SWELL1 is an essential VRAC component. These findings enable further molecular characterization of the VRAC channel complex and genetic studies for understanding the function of VRAC in normal physiology and disease.

钾与信号抑制剂对外生菌根真菌分泌乙酸的调控作用

PDF HTML阅读 XML下载 导出引用 引用提醒 钾与信号抑制剂对外生菌根真菌分泌乙酸的调控作用 DOI: 10.5846/stxb201307091871 作者: 作者单位: 西南大学资源环境学院,西南大学资源环境学院,西南大学资源环境学院,西南大学资源环境学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金（40771112，41171215）；中央高校基本科研业务费专项资金资助（XDJK2014C105，XDJK2012C035）；西南大学博士基金项目（SWU112083）；西南大学研究生科技创新基金项目（Ky2009022） Regulation of potassium supply and signal inhibitors on acetate effluxes by ectomycorrhizal fungi Author: Affiliation: College of Resources Environment,Southwest University,Beibei,College of Resources Environment,Southwest University,Beibei,College of Resources Environment,Southwest University,Beibei,College of Resources Environment,Southwest University,Beibei Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:外生菌根真菌是森林生态系统中的重要成分，参与树木养分的吸收利用。试验液体培养外生菌根真菌，设置不同供钾水平，添加钙信号抑制剂，研究了它们的有机酸和氢离子分泌，以及乙酸分泌对供钾和信号抑制剂的响应。结果表明，供试菌株的生长，氮、磷、钾含量和吸收量因菌株不同而异，生物量变化于52.91-121.72 mg/瓶之间，相差1倍以上。外生菌根真菌吸收养分的差异可能与它们对土壤养分环境的长期适应、进化、选择有关。在外生菌根真菌的培养液中，分别检测出草酸、乙酸、苹果酸、柠檬酸和丁二酸等，以及大量的氢离子，说明菌根真菌能分解土壤含钾矿物，释放钾离子，改善寄主植物的钾营养。其中，乙酸分泌量较大，具有普遍性，低钾刺激分泌乙酸，高钾时分泌减少，其分泌速率与供钾浓度和菌丝吸钾量之间呈显著负相关（r=-0.734，r=-0.617，n=60）。钾对菌根真菌分泌乙酸的调控作用具有改善森林钾素营养，防止土壤养分淋失的生理和生态意义。此外，在低钾条件下，阴离子通道和钙信号抑制剂抑制外生菌根真菌分泌乙酸。说明钙信号和阴离子通道参与了乙酸分泌，缺钾可能是刺激乙酸分泌的原初信号，通过信号转导和一系列级联反应促进乙酸分泌。 Abstract:Potassium (K), an element required slightly lower than nitrogen (N), is one of the essential major nutrients for trees in forests. K deficiency inhibits tree growth and limits forest productivities. Ectomycorrhizal fungi are one of important microorganisms in forests. Many forest trees have evolved mutualistic symbioses with the fungi that contribute to their nutrition and growth. In the fungus-tree associations, the fungi obtain carbohydrates from host trees and, in turn, provide the plants with mineral nutrients such as phosphorus (P), calcium (Ca), magnesium (Mg) and K. In our present experiment, ectomycorrhizal fungi isolated from pine and eucalyptus forests were grown in liquid culture media with various K supplies (26, 130, and 650 mg/L K2SO4) and with Ca2+ signal and anion channel inhibitors such as Trifluoperazine, TFP; Verapamil, VP; Ruthenium red, RR and Niflumic acid, NIF. The efflux of acetate and protons by the fungal isolates in the response to the K supplies and inhibitors were studied in vitro in order to understand the mechanisms of ectomycorrhizal fungi to improve the nutrition of host plants. The results indicated that the fungal species varied greatly in both growth and nutrient absorption (N, P and K). The hyphae biomass at harvest, for example, showed at least twice difference, ranging from 52.91 mg/flask to 121.72 mg/flask. The reasons for the variations in growth and nutrition could be explained by the biological adaption to soil nutrient environments where they lived for a long time through evolution and selection. Oxalate, acetate, malate, citrate, succinate and large amount of protons were detected in the culture solutions. Taking into the account of dissolution and decomposition of minerals and rocks caused by protons and organic acids, ectomycorrhizal fungi could thus make K in mineral structures available for the host plants. In addition, all study fungi released acetate into liquid culture media and the low K supply stimulated significantly the acetate efflux. The efflux rate correlated negatively with K concentration in culture solutions (r=-0.734, n=60) and fungal K absorption (r=-0.617, n=60). In forest soils, therefore, the influence of K on acetate efflux by the external hyphae of ectomycorrhizas could be physiologically and ecologically important for the improvement of tree K nutrition at low K supply but prevention against K leaching in fertile soils. The inhibitors of Ca signals and anion channels decreased the acetate efflux by ectomycorrhizal fungal isolates in culture solutions with low K supply. It seems reasonable to suggest both Ca signals and anion channels involved the process of the fungal acetate exudation in this case. The primary signal of K deficiencies could act as the intracellular second messenger to react on calmodulin (CaM), by which changed the Ca2+ distribution inside and outside of the hyphae cells, and then stimulated the cascade reactions responsible for acetate efflux, including gene expression, acetate synthesis and anion channel activation. Concerning the difference between the ectomycorrhizas in the field and the fungal isolates in vitro, further investigation is necessary to carry out on the mechanisms of the detail processes with ectomycorrhizal trees. 参考文献 相似文献 引证文献

Structural and functional dynamics of Excitatory Amino Acid Transporters (EAAT)

Glutamate transporters control the glutamate homeostasis in the central nervous system, and, thus, are not only crucial for physiological excitatory synaptic signaling, but also for the prevention of a large number of neurodegenerative diseases that are associated with excessive and prolonged presence of the neurotransmitter glutamate in the extracellular space. Until now, five subtypes of high-affinity glutamate transporters (excitatory amino acid transporters, EAATs 1-5) have been identified. These 5 high-affinity glutamate transporter subtypes belong to the solute carrier 1 (SLC1) family of transmembrane proteins: EAAT1/GLAST (SLC1A3), EAAT2/GLT1 (SLC1A2), EAAT3/EAAC1 (SLC1A1), EAAT4 (SLC1A6) and EAAT5 (SLC1A7). EAATs are secondary-active transporters, taking up glutamate into the cell against a substantial concentration gradient. The driving force for concentrative uptake is provided by the co-transport of Na + ions and the counter-transport of one K + in a step independent of the glutamate translocation step. Due to the electrogenicity of transport, the transmembrane potential can also act as driving force. Glutamate transporters are also able to run in reverse, resulting in glutamate release from cells. Due to these important physiological functions, glutamate transporter expression and, therefore, the transport rate, are tightly regulated. The EAAT protein family are structurally expected to be highly similar, however, these transporters show a functional diversity that ranges from high capacity glutamate uptake systems (EAATs 1-3) to receptor-like glutamate activated anion channels (EAATs 4-5). Here, we provide an update on most recent progress made on EAAT's molecular transport mechanism, structure-function relationships, pharmacology, and will add recent insights into mechanism of rapid membrane trafficking of glutamate transporters.

High glutamate permeability and distal localization of Best1 channel in CA1 hippocampal astrocyte

BackgroundGlutamate is the major neurotransmitter that mediates a principal form of excitatory synaptic transmission in the brain. From the presynaptic terminals of neurons, glutamate is released upon exocytosis of the glutamate-packaged vesicles. In recent years, astrocytes are also known to release glutamate via various routes to modulate synaptic transmission. In particular, we have characterized a glutamate-permeable Ca2+-activated anion channel encoded by Bestrophin 1 gene (Best1) that is responsible for Ca2+-dependent, channel-mediated glutamate release in astrocyte. Best1 channel contains a large pore that is readily permeable to large molecules such as glutamate and GABA. In those studies we obtained permeability ratio of glutamate to Cl- in heterologously expressed mouse Best1 in HEK293T cells and in endogenously expressed mouse Best1 in cultured astrocytes. However, up to now, glutamate permeability of the native Best1 channel in vivo has not been reported.FindingsIn whole-cell recordings of CA1 hippocampal astrocytes, we found that opening of Best1 channel upon activation of a Gq-coupled GPCR, protease-activated receptor 1 (PAR1) generated the anion current carried by glutamate via Ca2+ increase. This Ca2+-evoked glutamate-mediated anion current was unaffected by pretreatment of the inhibitors for a gap junction hemi-channel or Ca2+-activated K+ conductance. This astrocytic anion conductance carried by glutamate was mediated by Best1 channel expression in CA1 hippocampal astrocytes, because Best1 knock-down by shRNA expression eliminated astrocytic glutamate conductance by PAR-1 activation. However, we found that these astrocytes showed a deviation in reversal potential of Best1-mediated current from the predicted value. By performing dual patch recording, we concluded that the deviation of reversal potential is due to incomplete space clamping arising from extremely leaky membrane (input resistance ranging 1–3 MΩ), very low length constant of astrocytic processes, and the localization of Best1 channel in distal microdomains near synapses. Based on the relative shift of reversal potentials by ion substitutions, we estimated the permeability ratio of glutamate and Cl- (Pglutamate/PCl) as 0.53.ConclusionsOur study shows that Best1, located at the microdomains near the synaptic junctions, has a significantly high permeability to glutamate in vivo, serving as the prominent glutamate-releasing channel in astrocytes, mediating the release of various gliotransmitters in the brain, and playing an important role in modulating synaptic transmission.

Flow‐limiting valve for ABA signalling in stomatal guard cells

Flow‐limiting valve for <scp>ABA</scp> signalling in stomatal guard cells

Putative pore‐loops of TMEM16/anoctamin channels affect channel density in cell membranes

The recently identified TMEM16/anoctamin protein family includes Ca(2+)-activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5-TM6 flanking a re-entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca(2+) binding. The role of the putative pore-loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole-cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca(2+). Furthermore, whole-cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B-mediated currents. Replacement of the putative pore-loop of TMEM16A with that of TMEM16B (TMEM16A-B channels) reduced the currents by ∼six-fold, while the opposite modification (TMEM16B-A channels) produced a ∼six-fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca(2+) or voltage, nor were they due to changes in single-channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore-loop of TMEM16A and TMEM16B led to the identification of a region containing a non-canonical trafficking motif. Chimeras composed of the putative pore-loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore-loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for the putative pore-loop of TMEM16A and TMEM16B channels in the control of the whole-cell Ca(2+)-activated Cl(-) conductance.

PYR/RCAR Receptors Contribute to Ozone-, Reduced Air Humidity-, Darkness-, and CO2-Induced Stomatal Regulation

Rapid stomatal closure induced by changes in the environment, such as elevation of CO2, reduction of air humidity, darkness, and pulses of the air pollutant ozone (O3), involves the SLOW ANION CHANNEL1 (SLAC1). SLAC1 is activated by OPEN STOMATA1 (OST1) and Ca(2+)-dependent protein kinases. OST1 activation is controlled through abscisic acid (ABA)-induced inhibition of type 2 protein phosphatases (PP2C) by PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR) receptor proteins. To address the role of signaling through PYR/RCARs for whole-plant steady-state stomatal conductance and stomatal closure induced by environmental factors, we used a set of Arabidopsis (Arabidopsis thaliana) mutants defective in ABA metabolism/signaling. The stomatal conductance values varied severalfold among the studied mutants, indicating that basal ABA signaling through PYR/RCAR receptors plays a fundamental role in controlling whole-plant water loss through stomata. PYR/RCAR-dependent inhibition of PP2Cs was clearly required for rapid stomatal regulation in response to darkness, reduced air humidity, and O3. Furthermore, PYR/RCAR proteins seem to function in a dose-dependent manner, and there is a functional diversity among them. Although a rapid stomatal response to elevated CO2 was evident in all but slac1 and ost1 mutants, the bicarbonate-induced activation of S-type anion channels was reduced in the dominant active PP2C mutants abi1-1 and abi2-1. Further experiments with a wider range of CO2 concentrations and analyses of stomatal response kinetics suggested that the ABA signalosome partially affects the CO2-induced stomatal response. Thus, we show that PYR/RCAR receptors play an important role for the whole-plant stomatal adjustments and responses to low humidity, darkness, and O3 and are involved in responses to elevated CO2.

Acid-sensitive outwardly rectifying (ASOR) anion channels in human epithelial cells are highly sensitive to temperature and independent of ClC-3

A novel type of anion channel activated by extracellular acidification, called acid-sensitive outwardly rectifying (ASOR) anion channel, was shown to be involved in acidotoxic necrotic death in human epithelial cells. However, its biophysical property and molecular identity have remained elusive. In human epithelial HeLa cells, here, whole-cell currents of ASOR anion channel were found to be augmented by warm temperature, with a threshold temperature of 32°C. Temperature sensitivity of the conductance was found to be high (with Q 10 of 8.8) in the range of body temperature, suggesting a possible involvement of a non-diffusion-limited process such as a transporter-mediated conduction. In this regard, it is interesting that a Cl(-)/H(+) antiporter ClC-3 has recently been proposed as a candidate for the ASOR channel. However, siRNA-mediated knockdown of hClC-3 failed to suppress ASOR currents in HeLa cells. Also, endogenous ASOR currents in HEK293T cells were not affected by overexpression of human or mouse ClC-3. Furthermore, functional expression of the ASOR channel was virtually absent in the cisplatin-resistant human cancer KCP-4 cell line despite the fact that molecular expression of ClC-3 was indistinguishable between KCP-4 cells and parental cisplatin-sensitive KB-3-1 cells which endogenously exhibit high activity of ASOR anion channels. These results indicate that the ASOR anion channel is highly sensitive to temperature and independent of ClC-3.

Spontaneous mutation 7B-1 in tomato impairs blue light-induced stomatal opening

It was reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L.), an ABA overproducer, is defective in blue light (BL) signaling leading to BL-specific resistance to abiotic and biotic stresses. In this work, we examine responses of stomata to blue, red and white lights, fusicoccin, anion channel blockers (anthracene-9-carboxylic acid; 9-AC and niflumic acid; NIF) and ABA. Our results showed that the aperture of 7B-1 stomata does not increase in BL, suggesting that 7B-1 mutation impairs an element of BL signaling pathway involved in stomatal opening. Similar stomatal responses of 7B-1 and wild type (WT) to fusicoccin or 9-AC points out that activity of H+-ATPase and 9-AC-sensitive anion channels per se is not likely affected by the mutation. Since 9-AC restored stomatal opening of 7B-1 in BL, it seems that 9-AC and BL could block similar type of anion channels. The stomata of both genotypes did not respond to NIF neither in darkness nor in any light conditions tested. In light, 9-AC but not NIF restored stomatal opening inhibited by ABA in WT and 7B-1. We suggest that in comparison to WT, the activity of S-type anion channels in 7B-1 is more promoted by increased ABA content, and less reduced by BL, because of the mutant resistance to BL.

TMEM16F (Anoctamin 6), an anion channel of delayed Ca2+ activation

Members of the TMEM16 (Anoctamin) family of membrane proteins have been shown to be essential constituents of the Ca2+-activated Cl− channel (CaCC) in many cell types. In this study, we have investigated the electrophysiological properties of mouse TMEM16F. Heterologous expression of TMEM16F in HEK293 cells resulted in plasma membrane localization and an outwardly rectifying ICl,Ca that was activated with a delay of several minutes. Furthermore, a significant Na+ current was activated, and the two permeabilities were correlated according to PNa = 0.3 PCl. The current showed an EC50 of 100 µM intracellular free Ca2+ concentration and an Eisenman type 1 anion selectivity sequence of PSCN > PI > PBr > PCl > PAsp. The mTMEM16F-associated ICl,Ca was abolished in one mutant of the putative pore region (R592E) but retained in two other mutants (K616E and R636E). The mutant K616E had a lower relative permeability to iodide, and the mutant R636E had an altered anion selectivity sequence (PSCN = PI = PBr = PCl > PAsp). Our data provide evidence that TMEM16F constitutes a Ca2+-activated anion channel or a pore-forming subunit of an anion channel with properties distinct from TMEM16A.

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

Open stomata 1 (OST1) kinase controls R–type anion channel QUAC1 in Arabidopsis guard cells

Under drought stress, the stress hormone ABA addresses the SnR kinase OST1 via its cytosolic receptor and the protein phosphatase ABI1. Upon activation, OST1 phosphorylates the guard cell S-type anion channel SLAC1. Arabidopsis ABI1 and OST1 loss-of-function mutants are characterized by an extreme wilting 'open stomata' phenotype. Given the fact that guard cells express both SLAC- and R-/QUAC-type anion channels, we questioned whether OST1, besides SLAC1, also controls the QUAC1 channel. In other words, are ABI1/OST1 defects preventing both of the guard cell anion channel types from operating properly in terms of stomatal closure? The activation of the R-/QUAC-type anion channel by ABA signaling kinase OST1 and phosphatase ABI1 was analyzed in two experimental systems: Arabidopsis guard cells and the plant cell-free background of Xenopus oocytes. Patch-clamp studies on guard cells show that ABA activates R-/QUAC-type currents of wild-type plants, but to a much lesser extent in those of abi1-1 and ost1-2 mutants. In the oocyte system the co-expression of QUAC1 and OST1 resulted in a pronounced activation of the R-type anion channel. These studies indicate that OST1 is addressing both S-/SLAC- and R-/QUAC-type guard cell anion channels, and explain why the ost1-2 mutant is much more sensitive to drought than single slac1 or quac1 mutants.

Drosophila Bestrophin-1 Currents Are Regulated by Phosphorylation via a CaMKII Dependent Mechanism

Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca2+. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca2+/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.

Channel-mediated astrocytic glutamate release via Bestrophin-1 targets synaptic NMDARs

BackgroundAstrocytes regulate neuronal excitability and synaptic activity by releasing gliotransmitters such as glutamate. Our recent study demonstrated that astrocytes release glutamate upon GPCR activation via Ca2+ activated anion channel, Bestrophin-1 (Best1). The target of Best1-mediated astrocytic glutamate has been shown to be the neuronal NMDA receptors (NMDAR). However, whether it targets synaptically or extra-synaptically localized NMDAR is not known.FindingsWe recorded spontaneous miniature excitatory postsynaptic currents (mEPSCs) from CA1 pyramidal cells to test whether Best1-mediated astrocytic glutamate targets synaptic NMDAR. An agonist of protease activated receptor 1 (PAR1) was used to induce astrocytic Ca2+ increase and glutamate release. Firstly, we found that activation of PAR1 and subsequent release of glutamate from astrocyte does not alone increase the frequency of mEPSCs. Secondly, we found that mEPSC rise time is variable depending on the different electrotonic distances from the somatic recording site to the synaptic region where each mEPSC occurs. Two subgroups of mEPSC from CA1 pyramidal neuron by rise time were selected and analyzed. One group is fast rising mEPSCs with a rise time of 1 ~ 5 ms, representing synaptic activities arising from proximal dendrites. The other group is slowly rising mEPSCs with a rise time of 5 ~ 10 ms, representing synaptic events arising from glutamate release at synapses located in the distal dendrites. We used cell-type specific Best1 gene silencing system by Cre-loxP cleavage to dissociate the effect of neuronal and astrocytic Best1. Astrocytic Best1-mediated glutamate release by PAR1 activation did not affect decay kinetics, frequency, and amplitude of fast rising mEPSC. In contrast, PAR1 activation resulted in an NMDA receptor component to be present on slowly rising mEPSC, but did not alter frequency or amplitude.ConclusionsOur results indicate that astrocytic glutamate via Best1 channel targets and activates synaptic NMDARs.

New mechanism for glutamate hypothesis in epilepsy

Epilepsy is a broad range of neurological conditions that are manifested as seizures. Two major hypotheses—glutamate and potassium—have been proposed for the mechanism of epilepsy development (Fisher et al., 1976; During and Spencer, 1993). Although both hypotheses have some evidence to support, the relative contribution of potassium and glutamate to epilepsy has not been determined. Glutamate is a major excitatory neurotransmitter in the brain and an immediate precursor for GABA in neurons and glutamine in astrocytes. Glutamate is differentially compartmentalized and metabolized via different enzymes by astrocytes and neurons and exogenous and endogenous glutamate is handled distinctively by them (McKenna, 2007). Elevated levels of glutamate have been reported in human brain tissues and animal models of epilepsy, and it is known that glutamate-induced excitotoxicity causes the neuronal death in epilepsy (Haglid et al., 1994; Coulter and Eid, 2012 for detail). The glutamate-glutamine cycle is a major recycling mechanism of glutamate and GABA in the brain. A glutamate degrading enzyme, glutamine synthetase (GS) has been shown to be deficient in astrocytes in the epileptogenic hippocampal formation in a subset of patients with temporal lobe epilepsy (TLE) (Eid et al., 2004). This GS deficiency leads to increased glutamate levels in astrocytes as well as elevated concentrations of extracellular glutamate. Rats chronically infused with methionine sulfoximine, a GS inhibitor, showed increased glutamate in astrocytes (Perez et al., 2012). Increased glutamate release from neurons and astrocytes and/or impaired removal of glutamate in the extra-cellular space (e.g., synaptic cleft) could raise glutamate level. Glutamate clearance is mainly performed by glutamate transporters that move glutamate and potassium across the plasma membrane (Had-Aissouni, 2012). The astroglial sodium-dependent glutamate transporter-1 (GLT-1 a.k.a. EAAT2) is the major glutamate uptake molecule in the brain, and either malfunction and/or down-regulation of GLT-1 could cause elevated glutamate level. The fact that GLT-1 null mice are epileptic supports GLT-1 involvement for increased glutamate (Tanaka et al., 1997). However, findings of GLT-1 level in animal models and human TLE are not consistent, and they do not seem to fully explain the elevated glutamate (Mathern et al., 1999; Crino et al., 2002; Proper et al., 2002; van der Hel et al., 2005). It is evident that astrocytes are potential sources of the excessive glutamate in TLE, however, the mechanism(s) for glutamate release from astrocytes has been controversial; either vesicular exocytosis or channel/transporter-mediated and whether calcium is involved or not (Tian et al., 2005). A recent study demonstrating two different modes of glutamate release in astrocytes provides a new way of thinking for epilepsy: TREK-1, a two-pore potassium channel, can be responsible for fast glutamate release induced by GPCR (G-protein coupled receptors) activation and Bestrophin-1 (Best1), a calcium activated anion channel for slow release (Woo et al., 2012). To detect released glutamate from astrocytes, a technique called “sniffer-patch” was used: electrophysiological recording of HEK293T cells expressing a non-desensitizing mutant (GluR1-L497Y) of AMPA receptors were co-cultured with dissociated hippocampal astrocytes. An agonist peptide for PAR1 (protease-activated receptor 1) was applied to activate G-protein in astrocytes. Thus, glutamate released from astrocytes can be measured via AMPA receptor currents. Differential subcellular localization of these two channels also raises the possibility of distinct mode of their operations in epilepsy. TREK-1 is preferentially expressed at cell bodies and processes of astrocytes, while Best1 is present close to synapses. It remains to be seen whether their expression and localization in the brain is altered in epilepsy. The involvement of TREK-1, which was identified as a potassium channel, here is particularly interesting. The potassium hypothesis of epilepsy was proposed about a half century ago (Green, 1964; Fisher et al., 1976) and inactivation mutations of several potassium channels cause human and rodent epilepsies (Benarroch, 2009). Impaired spatial potassium buffering by astrocytes will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent potassium release, and may thus contribute to seizure generation in this particular condition of human TLE (Hinterkeuser et al., 2000). TREK-1 null mice have increased seizure susceptibility to systemic kainate administration. Is the pore of TREK-1 permeable both to potassium and glutamate? Is there any selective mechanism of TREK-1 for one molecule over the other depending on the activating signals? The answers to these questions may aid in dissecting the relative contribution of these two molecules to hyperexcitability and epilepsy. As for Best1, the question is whether enhanced calcium signals in astrocytes directly affect Best1's function in epilepsy models (Heurteaux et al., 2004; Ding et al., 2007). Therefore, it will be very intriguing to examine the workings of TREK-1 and Best1 in animal models and human TLE.

Neuroprotective effects of volume-regulated anion channel blocker DCPIB on neonatal hypoxic-ischemic injury

To evaluate the role of swelling-induced activation of volume-regulated anion channels (VRACs) in a neonatal hypoxic-ischemic injury model using the selective VRAC blocker 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on5-yl) oxobutyric acid (DCPIB). Cerebral hypoxic-ischemic injury was induced in 7-day-old mouse pups with Rice-Vannucci method. Prior to the onset of ischemia, the animals were ip administered DCPIB (10 mg/kg). The animals were sacrificed 24 h afterwards, coronal sections of the brains were cut and the areas of infarct were examined using TTC staining and an image-analysis system. Cultured PC12 cells were subjected to oxygen-glucose deprivation (OGD) for 4 h. The cellular viability was assessed using Cell Counting Kit 8. Intracellular chloride concentration [Cl(-)](i) was measured using 6-methoxy-N-ethylquinolinium iodide. DCPIB-treated mice showed a significant reduction in hemispheric corrected infarct volume (26.65%±2.23%) compared to that in vehicle-treated mice (45.52%±1.45%, P<0.001). DCPIB-treated mice also showed better functional recovery as they were more active than vehicle-treated mice at 4 and 24 h post injury. In cultured PC12 cells, DCPIB (10 μmol/L) significantly reduced OGD-induced cell death. Moreover, DCPIB (20 μmol/L) blocked hypotonic-induced decrease in [Cl(-)](i) in PC12 cells of both control and OGD groups. The results further support the pathophysiological role of VRACs in ischemic brain injury, and suggest DCPIB as a potential, easily administrable agent targeting VRACs in the context of perinatal and neonatal hypoxic-ischemic brain injury.

Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis

BackgroundGlutamate is the major transmitter that mediates the principal form of excitatory synaptic transmission in the brain. It has been well established that glutamate is released via Ca2+-dependent exocytosis of glutamate-containing vesicles in neurons. However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.FindingsWe report a novel form of glutamate release in astrocytes via the recently characterized Ca2+-activated anion channel, Bestrophin-1 (Best1) by Ca2+ dependent mechanism through the channel pore. We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.ConclusionsThese results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

A role for oxalic acid generation in ozone‐induced signallization in Arabidopis cells

Ozone (O(3) ) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O(3) results in ascorbate-derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca(2+) concentration, generation of reactive oxygen species and cell death. We confirmed that O(3) reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O(3) -induced signalling pathways that trigger programmed cell death.

A G Protein Blocks the Chilling Effect

Activation of an ion channel that responds to cold temperature is inhibited by the direct association of a G protein α subunit.

TREK-1 and Best1 Channels Mediate Fast and Slow Glutamate Release in Astrocytes upon GPCR Activation

Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(βγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(βγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 μM, whereas slow mode targets neuronal NMDA receptors at around 1 μM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.