Electronic Theses and Dissertations

Identifier

1188

Date

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Concentration

Organic Chemistry

Committee Chair

Abby L. Parrill

Committee Member

Daniel L. Baker

Committee Member

Theodore J. Burkey

Committee Member

Richard L. Petersen

Abstract

The large-conductance, voltage- and calcium-activated potassium channel (BKCa channel) is an important transmembrane ion channel involved in many physiological and pathophysiological processes. However, little is known about its atomic-level three-dimensional structure. The functional channel requires four alpha subunits assembled as a potassium pore and is typically associated with four beta subunits that regulate function in various ways depending on the isoform of the beta subunit is interacting with the channel. Currently, the only major high-resolution atomic structure available is of the cytosolic tail domain of the alpha subunit. In the absence of structural information, electrophysiology has allowed for extensive characterization of the channel’s role in physiological processes and advanced understanding of BKCa channel pharmacology including inhibition of BKCa by physiologically relevant concentrations of cholesterol and stimulation of BKCa by the related sterol, lithocholic acid. However, many questions still remain including how does cholesterol exert effects on the alpha subunit, what are the structures of the different beta subunits, and can therapeutic lead compounds be developed to selectively target BKCa beta subunits in a tissue specific manner. The difficulty of obtaining transmembrane protein structures has made characterization a major challenge.Herein, work is described where 1) the BKCa beta 1 subunit was successfully expressed and purified from E. coli for the first time with a yield of approximately 32mg/L of bacterial growth and several assays were attempted to demonstrate protein functionality. However, functional assessment of purified protein has remained elusive due to the hydrophobic nature of known ligands. Additional effort is needed to establish ligand recognition using solution nuclear magnetic resonance spectroscopy. 2) Multiple generations of pharmacophore models have been developed to aid in virtual screening efforts to find therapeutic lead compounds targeting BKCa through the beta 1 subunit. Sixteen compounds have been selected from virtual screening of the PubChem database that can be tested in vitro for model validation. 3) Molecular dynamics simulations were utilized to study the underlying mechanism of the interaction between cholesterol and the BKCa alpha subunit cytosolic tail domain showing differential behavior of cholesterol in mutant and wild type simulations that agree with experimental data

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.

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