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, alpha ENaC, and gamma 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 acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, alpha ENaC, gamma ENaC, and delta ENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, alpha ENaC, or gamma 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 alpha ENaC or gamma ENaC also abolished the high P-K(+)/P-Na(+) of D54-MG cells. Knocking down gamma ENaC in D54-MG cells reduced delta ENaC protein expression but had no effect on either the whole cell current or K+ permeability. Using coimmunoprecipitation we show interactions between ASIC1, alpha ENaC, and gamma 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, alpha ENaC, or gamma ENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.