title: 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, alphaENaC, and gammaENaC 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, alphaENaC, gammaENaC, and deltaENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, alphaENaC, or gammaENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down alphaENaC or gammaENaC also abolished the high P(K)(+)/P(Na)(+) of D54-MG cells. Knocking down deltaENaC in D54-MG cells reduced deltaENaC protein expression but had no effect on either the whole cell current or K(+) permeability. Using co-immunoprecipitation we show interactions between ASIC1, alphaENaC, and gammaENaC, 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, alphaENaC, or gammaENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.
authors: Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors: Niren
authors: N
authors: Kapoor
authors: Rafal
authors: R
authors: Bartoszewski
authors: Yawar J
authors: YJ
authors: Qadri
authors: Zsuzsanna
authors: Z
authors: Bebok
authors: James K
authors: JK
authors: Bubien
authors: Catherine M
authors: CM
authors: Fuller
authors: Dale J
authors: DJ
authors: Benos
mesh_terms: D062565:Acid Sensing Ion Channels; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D016466:CHO Cells; D002465:Cell Movement; D006224:Cricetinae; D003412:Cricetulus; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005909:Glioblastoma / Q000235:genetics / Q000401:mortality*; D006801:Humans; D008564:Membrane Potentials; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: C568266:ASIC1 protein, human; D062565:Acid Sensing Ion Channels; D053503:Epithelial Sodium Channels; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D015222:Sodium Channels