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

Kapoor N, Lee W, Clark E, Bartoszewski R, McNicholas CM, Latham CB, Bebok Z, Parpura V, Fuller CM, Palmer CA, Benos DJ. Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes. Am J Physiol Cell Physiol 300: C1246-C1259, 2011. First published February 23, 2011; doi: 10.1152/ajpcell.00199.2010.-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 alpha ENaC and gamma ENaC 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 alpha ENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus gamma ENaC-YFP. TIRFM images show colocalization of ASIC1 with both alpha ENaC and gamma ENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and alpha ENaC 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 approximate to 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.