All ETDs from UAB

Advisory Committee Chair

Dale J Benos

Advisory Committee Members

John J Hablitz

Robin A Lester

Lori L McMahon

Anne B Theibert

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Glioblastoma Multifrome 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, 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 Epithelial Sodium Channel (ENaC) and Acid Sensing Ion Channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, and γENaC. D54-MG glioma cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression for each of the specific subunits and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. We also show an interaction between ASIC1, αENaC, and γENaC on the plasma membrane of D54-MG glioma cells using co-immunoprecipitation and in transfected rat primary astrocytes using Total Internal Reflection Fluorescence (TIRF) microscopy. Knocking down ASIC1, αENaC, or γENaC also significantly inhibited glioma cell migration compared to cells without subunit knockdown consistent with the hypothesis that these subunits play an important role in glioma biology. Using immunohistochemisty we show a higher expression level of ASIC1, αENaC, and γENaC in GBM biopsy tissue compared with non-neoplastic brain tissue. To understand the reason for this increase in expression of ASIC1 and ENaC subunits in GBM cells and tissue compared with primary astrocytes and non-neoplastic brain tissue, we used TIRF microscopy to image live glioma cells under acidic conditions (pH 6.0), similar to the acidic environment found in the core of a glioblastoma lesion.We found an increase in the plasma membrane localization of ASIC1 in D54-MG glioma cells when cells were grown at pH 6.0 compared to pH 7.4. Using High Resolution Clear Native electrophoresis we also found that ASIC1 forms a complex with ENaC subunits in D54-MG glioma cells that migrate at ≈ 480 kDa. These data suggest that different ENaC/Deg subunits can combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.

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