Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test the hypothesis that ASIC1 interacts with αENaC and γENaC at the cellular level, we have used total internal reflection fluorescence microscopy (TIRFM) in live rat astrocytes transiently cotransfected with cDNAs for ASIC1-DsRed plus αENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus γENaC-YFP. TIRFM images show colocalization of ASIC1 with both αENaC and γENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and αENaC both localize to a submembrane region following exposure to pH 6.0, similar to the acidic conditions found in the core of a glioblastoma lesion. Using high-resolution clear native gel electrophoresis, we found that ASIC1 forms a complex with ENaC subunits which migrates at ≈480 kDa in D54-MG glioma cells. These data suggest that different ENaC/Deg subunits interact and could combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.     

Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.     

Participation of the chaperone Hsc70 in the trafficking and functional expression of ASIC2 in glioma cells

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.     

Glioma-specific cation conductance regulates migration and cell cycle progression

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21(Cip1) and p27(Kip1). Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaC in D54MG cells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas.     

IL-10 and TGF-β2 are overexpressed in tumor spheres cultured from human gliomas

Immune-associated cytokines including IL-10 and TGF-β2 are thought to play a crucial role in immunosuppression mediated by gliomas. We have investigated the possibility that glioma stem cells are the major source of these cytokines. Tumor spheres, clonal non-adherent cell colonies derived from a single tumor stem cell, were cultured from surgical specimens of eight glioma patients, including two glioblastoma multiformes (grade IV), one anaplastic oligodendroglioma (grade III) and five anaplastic astrocytomas (grade III). Real-time RT-PCR and immunoassay were used to compare the relative expression levels of IL-10 and TGF-β2 in stem-cell-derived tumor sphere cells (TSCs) and primary cultured glioma cells (PCGCs). TSCs were confirmed to express the brain tumor stem cell marker CD133, and on in vitro differentiation gave rise to cells expressing neuronal or glial markers. RT-PCR and immunoassay revealed that mRNA and protein levels of both IL-10 and TGF-β2 were significantly higher in TSCs than in PCGCs from the same tumor. Interestingly, the degree of overexpression in TSCs, but not in PCGS, appeared to correlate with the pathological grade of the glioma. These findings suggest that glioma stem cells are likely to be the major tumor source of immunosuppressive cytokines and thereby play a crucial role in determining glioma malignancy.     

Identification of acid-sensing ion channels in adenoid cystic carcinomas

Tissue acidosis is an important feature of tumor. The response of adenoid cystic carcinoma (ACC) cells to acidic solution was studied using whole-cell patch-clamp recording in the current study. An inward, amiloride-sensitive Na(+) current was identified in cultured ACC-2 cells while not in normal human salivary gland epithelial cells. Electrophysiological and pharmacological properties of the currents suggest that heteromeric acid-sensing ion channels (ASICs) containing 2a and 3 may be responsible for the proton-induced currents in the majority of ACC-2 cells. Consistent with it, analyses of RT-PCR and Western blotting demonstrated the presences of ASIC2a and 3 in ACC-2 cells. Furthermore, we observed the enhanced expression of ASIC2a and 3 in the sample of ACC tissues. These results indicate that the functional expression of ASICs is characteristic feature of ACC cells.     

Proteolytic Cleavage of Human Acid-sensing Ion Channel 1 by the Serine Protease Matriptase

Acid-sensing ion channel 1 (ASIC1) is a H⁺-gated channel of the amiloride-sensitive epithelial Na⁺ channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases. The type II transmembrane serine protease matriptase activates the prototypical αβγENaC channel, but we found that matriptase is expressed in glioma cells and its expression is higher in glioma compared with normal astrocytes. Therefore, the goal of this study was to test the hypothesis that matriptase regulates ASIC1 function. Matriptase decreased the acid-activated ASIC1 current as measured by two-electrode voltage clamp in Xenopus oocytes and cleaved ASIC1 expressed in oocytes or CHO K1 cells. Inactive S805A matriptase had no effect on either the current or the cleavage of ASIC1. The effect of matriptase on ASIC1 was specific, because it did not affect the function of ASIC2 and no matriptase-specific ASIC2 fragments were detected in oocytes or in CHO cells. Three matriptase recognition sites were identified in ASIC1 (Arg-145, Lys-185, and Lys-384). Site-directed mutagenesis of these sites prevented matriptase cleavage of ASIC1. Our results show that matriptase is expressed in glioma cells and that matriptase specifically cleaves ASIC1 in heterologous expression systems.     

Amiloride-sensitive Na+ channels contribute to regulatory volume increases in human glioma cells

Despite intensive research, brain tumors remain among the most difficult type of malignancies to treat, due largely to their diffusely invasive nature and the associated difficulty of adequate surgical resection. To migrate through the brain parenchyma and to proliferate, glioma cells must be capable of significant changes in shape and volume. We have previously reported that glioma cells express an amiloride- and psalmotoxin-sensitive cation conductance that is not found in normal human astrocytes. In the present study, we investigated the potential role of this ion channel to mediate regulatory volume increase in glioma cells. We found that the ability of the cells to volume regulate subsequent to cell shrinkage by hyperosmolar solutions was abolished by both amiloride and psalmotoxin 1. This toxin is thought to be a specific peptide inhibitor of acid-sensing ion channel (ASIC1), a member of the Deg/ENaC superfamily of cation channels. We have previously shown this toxin to be an effective blocker of the glioma cation conductance. Our data suggest that one potential role for this conductance may be to restore cell volume during the cell's progression thorough the cell cycle and while the tumor cell migrates within the interstices of the brain.     

乳铁蛋白在人脑胶质瘤中的表达及意义

目的：通过对乳铁蛋白生物学特性的研究,分析其在人脑胶质瘤中的表达及意义。方法：通过免疫组化方法测定10例非肿瘤脑组织和46例胶质瘤（其中低级别胶质瘤27例（I级和II级）和高级别胶质瘤19例（III级和Ⅳ级）中乳铁蛋白的表达情况并分析二者的相关性。用RT—PCR技术及Westem—blot方法测定非肿瘤脑组织、低级别组胶质瘤和高级别胶质瘤组乳铁蛋白的表达量并分析二者的相关性。结果：乳铁蛋白在非肿瘤脑组织及不同级别胶质瘤中的表达情况不同,其在低级别胶质瘤中有表达,略低于非肿瘤脑组织;而在高级别胶质瘤中表达显著减少或者表达不明显,明显低于低级别胶质瘤和非肿瘤脑组织（免疫组化、RT．PCR及Western．Blot的P值分别为P=0．001、P=0．003、P=0．004）。结论：乳铁蛋白在非肿瘤脑组织和不同级别胶质瘤中的表达有差异,提示乳铁蛋白参与了人脑胶质瘤发生与发展的过程。高级别胶质瘤的高度恶性和高侵袭性可能与乳铁蛋白表达的下调从而丧失对肿瘤细胞的抑制作用相关。     

Epithelial sodium channel is regulated by SNAP-23/syntaxin 1A interplay

Sodium-selective amiloride-sensitive epithelial channel (ENaC) located in the apical membrane is involved in the reabsorption of sodium in tight epithelia. The soluble N-ethylmaleimide-sensitive attachment receptors (SNAREs) mediate vesicle trafficking in a variety of cell systems. Syntaxin (a t-SNARE) has been shown to interact with and functionally regulate a number of ion channels including ENaC. In this study, we investigated the role of SNAP-23, another SNARE protein, on ENaC activity in the HT-29 colonic epithelial cell system and Xenopus oocytes. Recording of amiloride-sensitive currents in both systems suggest that SNAP-23 modulates channel function, though a much higher concentration is required to inhibit ENaC in Xenopus oocytes. The introduction of Botulinum toxin A (a neurotoxin which cleaves SNAP-23), but not Botulinum toxin B or heat-inactivated Botulinum toxin A, reversed the inhibitory effect of SNAP-23 on amiloride-sensitive currents. However, syntaxin 1A and SNAP-23 combined portray a complex scenario that suggests that this channel interacts within a quaternary complex. Synaptotagmin expression neither interacts with, nor showed any effect on amiloride-sensitive currents when co-expressed with ENaC. Pull down assays suggest mild interaction between ENaC and SNAP-23, which gets stronger in the presence of syntaxin 1A. Data further suggest that SNAP-23 possibly interacts with the N-terminal alphaENaC. These functional and biochemical approaches provide evidence for a complex relationship between ENaC and the exocytotic machinery. Our data suggest that SNARE protein interplay defines the fine regulation of sodium channel function.     

Whole-cell currents in single and confluent M-1 mouse cortical collecting duct cells

M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl- Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M- 1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.     

Cation selectivity and inhibition of malignant glioma Na+ channels by Psalmotoxin 1

Psalmotoxin 1 (a component of the venom of a West Indies tarantula) is a 40-amino acid peptide that inhibits cation currents mediated by acid-sensing ion channels (ASIC). In this study we performed electrophysiological experiments to test the hypothesis that Psalmotoxin 1 (PcTX1) inhibits Na⁺ currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). In whole cell patch-clamped cultured GBM cells, the peptide toxin quickly and reversibly inhibited both inward and outward current with an IC₅₀ of 36 ± 2 pM. The same inhibition was observed in freshly resected GBM cells. However, when the same experiment was performed on normal human astrocytes, the toxin failed to inhibit the whole cell current. We also determined a cationic selectivity sequence for inward currents in three cultured GBM cell lines (SK-MG-1, U87-MG, and U251-MG). The selectivity sequence yielded a unique biophysical fingerprint with inward K⁺ conductance approximately fourfold greater than that of Na⁺, Li⁺, and Ca²⁺. These observations suggest that PcTX1 may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas. patch clamp; amiloride; ion channels; acid-sensing ion channels     

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

Abstract.  Objectives : Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca²⁺ influx pathway impacting cellular growth. Materials and Methods : Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results : We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl₃, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G₂+ M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions : Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme , the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours.     

Expression and Activity of Acid-Sensing Ion Channels in the Mouse Anterior Pituitary

Acid sensing ion channels (ASICs) are proton-gated cation channels that are expressed in the nervous system and play an important role in fear learning and memory. The function of ASICs in the pituitary, an endocrine gland that contributes to emotions, is unknown. We sought to investigate which ASIC subunits were present in the pituitary and found mRNA expression for all ASIC isoforms, including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. We also observed acid-evoked ASIC-like currents in isolated anterior pituitary cells that were absent in mice lacking ASIC1a. The biophysical properties and the responses to PcTx1, amiloride, Ca²⁺ and Zn²⁺ suggested that ASIC currents were mediated predominantly by heteromultimeric channels that contained ASIC1a and ASIC2a or ASIC2b. ASIC currents were also sensitive to FMRFamide (Phe-Met-Arg-Phe amide), suggesting that FMRFamide-like compounds might endogenously regulate pituitary ASICs. To determine whether ASICs might regulate pituitary cell function, we applied low pH and found that it increased the intracellular Ca²⁺ concentration. These data suggest that ASIC channels are present and functionally active in anterior pituitary cells and may therefore influence their function.     

Retracted: Superoxide‐dependent uptake of vitamin C in human glioma cells

Glioblastomas are lethal brain tumors that resist current cytostatic therapies. Vitamin C may antagonize the effects of reactive oxygen species (ROS) generating therapies; however, it is often used to reduce therapy‐related side effects despite its effects on therapy or tumor growth. Because the mechanisms of vitamin C uptake in gliomas are currently unknown, we evaluated the expression of the sodium‐vitamin C cotransporter (SVCT) and facilitative hexose transporter (GLUT) families in human glioma cells. In addition, as microglial cells can greatly infiltrate high‐grade gliomas (constituting up to 45% of cells in glioblastomas), the effect of TC620 glioma cell interactions with microglial‐like HL60 cells on vitamin C uptake (Bystander effect) was determined. Although glioma cells expressed high levels of the SVCT isoform‐2 (SVCT2), low functional activity, intracellular localization and the expression of the dominant‐negative isoform (dnSVCT2) were observed. The increased glucose metabolic activity of glioma cells was evident by the high 2‐Deoxy‐d ‐glucose and dehydroascorbic acid (DHA) uptake rates through the GLUT isoform‐1 (GLUT1), the main DHA transporter in glioblastoma. Co‐culture of glioma cells and activated microglial‐like HL60 cells resulted in extracellular ascorbic acid oxidation and high DHA uptake by glioma cells. This Bystander effect may explain the high antioxidative potential observed in high‐grade gliomas.

     

Malignant human gliomas express an amiloride-sensitive Na+ conductance

Human astrocytoma cells were studied using whole cellpatch-clamp recording. An inward, amiloride-sensitiveNa⁺ current was identified in fourcontinuous cell lines originally derived from human glioblastoma cells(CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures ofcells obtained from glioblastoma multiforme tumors (up to 4 passages).In addition, cells freshly isolated from a resected medulloblastomatumor displayed this same characteristic inward current. In contrast,amiloride-sensitive currents were not observed in normal humanastrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas.The only amiloride-sensitive Na⁺channels thus far molecularly identified in brain are the brain Na⁺ channels (BNaCs). RT-PCRanalyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 inthese tumors and in normal astrocytes. These results indicate that thefunctional expression of amiloride-sensitive Na⁺ currents is a characteristicfeature of malignant brain tumor cells and that this pathway may be apotentially useful target for therapeutic intervention.