Bruce Felts

Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Loren K. Henry

Abstract

The SLC6 family of secondary active transporters is made up of integral membrane solute carrier proteins characterized by the Na+-dependent translocation of small amino acid or amino acid-like substrates. SLC6 transporters, particularly the monoamine transporters (MATs) of serotonin, dopamine and norepinephrine, are some of the most heavily studied proteins today due to their association with a number of human diseases and disorders, making MATs a critical target for therapeutic development. In addition, MATs are directly involved in the action of drugs of abuse such as cocaine, amphetamines, and ecstasy.

Following the first cloning of a MAT gene in the early 1990s, much has been uncovered about the structure and function of these proteins. Early studies developed an understanding of the kinetic parameters by which MATs operate and also yielded enough information to model the basic structural characteristics of MATs. This was greatly improved upon within the last decade, as crystallographic and computational advances have provided structural insights that have vastly accelerated our ability to study these proteins and their involvement in complex biological processes. However, despite a wealth of knowledge concerning the structural and kinetic characteristics of MATs, little is understood as to how these features are interrelated and much is still unclear as to the how regulation (and maybe more importantly, dysregulation) of MATs alters the functionality of these proteins at the molecular and synaptic levels.

The overall goal of this dissertation was to comprehensively examine the relationship between MAT structure and the ions and ligands that bind to MATs to promote/prevent transporter function. This was done using a comprehensive approach that included biological, electrophysiological and computational techniques to target and elucidate the roles of specific amino acid residues in ion/ligand binding and/or mediation of the substrate translocation process. In successfully examining a number of specific MAT residues, this work has lead to the deduction of basic roles for each of the ion binding sites in the translocation mechanism (chapters II and III), as well as detailed the importance of specific structural components of MATs that are vital for functionality (chapters IV and V). Furthermore, this dissertation includes work highlighting the development of several photo-labeled, radio-iodinated antagonist analogues that will be used to further improve the understanding of how inhibitors bind to and block MAT function at the molecular level (chapter VI). In total, the work outlined in this dissertation provides a clearer understanding as to the molecular interactions that are necessary for MAT function and contributes an improved appreciation for the underlying mechanisms of substrate translocation and pharmacological intervention.

Download

Share

COinS