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

Abstract

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, ␣ENaC, and ␥ENaC 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 acidsensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, ␣ENaC, ␥ENaC, and ␦ENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, ␣ENaC, or ␥ENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down ␣ENaC or ␥ENaC also abolished the high P K ؉ /P Na ؉ of D54-MG cells. Knocking down ␦ENaC in D54-MG cells reduced ␦ENaC protein expression but had no effect on either the whole cell current or K ؉ permeability. Using coimmunoprecipitation we show interactions between ASIC1, ␣ENaC, and ␥ENaC, 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, ␣ENaC, or ␥ENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.

Glioma-specific Cation Conductance Regulates Migration and Cell Cycle Progression

“…The amiloride-sensitive cation conductance found in glioma cells is also blocked by psalmotoxin-1 (PcTX-1), a peptide isolated from West Indies tarantula Psalmopoeus cambridgei (17,18), and is a highly specific blocker of ASIC1. Knocking down expression of any of the three Deg/ ENaC subunits abolishes the current and slows migration (15). Furthermore, the regulatory volume increase of glioma cells following cell shrinkage by hyperosmolar solutions was completely abolished by PcTX-1 and amiloride (19).…”

“…Cells can also control their internal volume by regulating transport of Na ϩ , K ϩ , and Cl Ϫ across the plasma membrane, swelling or shrinking as required. Although much less sensitive to amiloride than the prototypical renal sodium channel ␣␤␥ENaC (K i amil Ͻ0.1 M (32)), the glioma cation channel that we have identified is critically involved in migration and proliferation (13)(14)(15)(16). This channel is inhibited by amiloride and its analogs and is blocked by PcTX-1, to which it is exquisitely sensitive (K i amil 10 -100 M; K i PcTX-1 K i ϳ40 pM (17)).…”

“…The cells were cultured and maintained in Dulbecco's modified Eagle's/ F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum (Thermo-Fisher) in the absence of antibiotics. To generate stable cell lines, D54-MG cells were transfected with 4 g of a truncated eGFP-ASIC1 or eGFP-␦ENaC cDNA as described previously (15). Following transfection, cells were cultured for 72 h, transferred to a T75 flask, and selected with G418 (500 g/ml; Mediatech, Manassas, VA).…”

“…This construct effectively knocks down expression of endogenous ASIC1, but it does not affect expression of other related subunits (15). We analyzed whole-cell lysates obtained from both wild type D54-MG (D54MG-WT) cells and D54-MG cells stably transfected with the DN-eGFP-ASIC1 cDNA (D54MG-A1DN) for ASIC-1 protein expression.…”

“…This cation current is due to expression of a cross-clade channel composed of members of the Deg/ENaC channel superfamily, i.e. acid-sensitive ion channel 1 (ASIC1) and two members of the epithelial sodium channel family, ␣-and ␥ENaC (13)(14)(15)(16). The amiloride-sensitive cation conductance found in glioma cells is also blocked by psalmotoxin-1 (PcTX-1), a peptide isolated from West Indies tarantula Psalmopoeus cambridgei (17,18), and is a highly specific blocker of ASIC1.…”