Niren Kapoor

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, αENaC, and γENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. 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 this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high PK+/PNa+ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K+ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, αENaC, or γENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.

The Unfolded Protein Response (UPR)-activated Transcription Factor X-box-binding Protein 1 (XBP1) Induces MicroRNA-346 Expression That Targets the Human Antigen Peptide Transporter 1 (TAP1) mRNA and Governs Immune Regulatory Genes

Background: The adaptive unfolded protein response (UPR) promotes endoplasmic reticulum (ER) expansion and reduces ER load. Results: UPR-activated XBP1 induces miR-346 expression that targets TAP1. Conclusion: We identify a novel function for XBP1 and an miRNA-mediated pathway for ER load reduction through TAP1. Significance: Novel interventions for protein folding disorders will require an understanding of how microRNAs regulate gene expression during ER stress. To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3′-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.

Heteromeric assembly of acid-sensitive ion channel and epithelial sodium channel subunits.

Amiloride-sensitive ion channels are formed from homo- or heteromeric combinations of subunits from the epithelial Na+ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.

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.

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1bs COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.