From gliomas to gorps: Role of the sodium(+)/proton exchanger in regulation of intracellular pH

The goal of my dissertation was to evaluate the role of the Na +/H+ exchanger (NHE) in the regulation of intracellular pH (pHi) in malignant gliomas. The ubiquitous NHE1 isoform is upregulated in gliomas as demonstrated by an alkaline shift in pH dependence compared to normal astrocytes. The importance of NHE to gliomas was demonstrated by inhibition studies, which resulted in pronounced acidification and cell death of gliomas with little effect on normal astrocytes. The elevated pHi and enhanced NHE1 activity was not the result of a mutant NHE isoform in the tumors, as demonstrated by DNA sequencing, indicating that alterations in NHE1 regulation must be involved. Although a variety of different genetic mutations may be involved in the pathogenesis of gliomas, including many steps in the growth factor signaling cascade, activation of NHE1 with corresponding intracellular alkalinization is a common feature of most of these tumors. This common phenotype makes the exchanger an attractive target for a tumor-selective therapy that should be effective regardless of the initial cause of the tumor pathogenesis.;To further my understanding of NHE function and regulation, I utilized the Amphiuma red blood cell (RBC) model system that has been well characterized by the Cala laboratory. The Amphiuma (a.k.a. "gorp") NHE is an attractive model due to its high abundance, low tonic activity, and robust response to both acidification and cell shrinkage, with kinetics that are remarkably similar to human NHE1 (hNHE1). The structure of the Amphiuma exchanger (atNHE1) is also highly homologous to hNHE1, with 79% identity and 89% similarity at the amino acid level. Furthermore, the proposed ion transport regions (M6--M7), inhibitor binding sites, and known regulatory domains are highly conserved between these two isoforms. Most importantly, the fact that the cloned atNHE1 protein is able to function in both pH and volume regulatory capacities, similar to that seen in native Amphiuma RBCs, indicates that the regulatory control mechanisms must be conserved between the two systems as well. Despite this remarkable homology, I found some significant functional differences between the human and Amphiuma exchangers which will provide further insight into the structure/function relationships of this ubiquitously expressed transporter.