Advisory Committee Chair
Lawrence J Delucas
Advisory Committee Members
Catherine M Fuller
Tim M Townes
Stephen G Aller
Thomas M Ryan
Champion Cs Deivanayagam
Document Type
Dissertation
Date of Award
2014
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Epithelial sodium channels (ENaC) play a critical role in maintaining Na+ homeostasis in various tissues throughout our body. Understanding of ENaC structure is mediated from studies of the homologous acid sensing ion channel 1 (ASIC1). However, ENaC has sev-eral notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elu-sive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric ENaC (α, ß, and γ), the α-subunit alone is of significant interest. αENaC is unique in that it is capable of forming a homo-trimeric structure capable of conducting Na+ ions. Despite functional and structural interest in αENaC, a key factor preventing structural studies of αENaC is its promiscuous interaction with several other proteins which disrupts its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC and increase its homogeniety. In the following we present a novel expression system coupled with a two-step affinity purification using NiNTA, followed by a GFP antibody column as a rapid procedure that provides relatively pure rat αENaC. The X-ray structural studies were carried out in two parts. First was the solving of crystallographic structure of Mycobacterium tuberculosis (TB) protein Rv3902c at 1.55Å resolution. While the function is not known, it is thought to be a virulence factor chaperone. This is due to it having structural similarity to the Salmonella virulence factor chaperone, InvB, and it is expressed on the same operon as Rv3903c, a known virulence factor. There is a hydrophobic pocket on the surface of the pro-tein that might be pivotal in its function as well as provide a potential drug target site. TB kills over 1 million people a year and this target site might provide a possi-ble treatment to TB virulence.
Recommended Citation
Reddy, Bharat G., "Epithelial Sodium Channel Purification And X-Ray Crystallographic Studies" (2014). All ETDs from UAB. 2803.
https://digitalcommons.library.uab.edu/etd-collection/2803