Attachment Sites Of Irreversible Cocaine Analogs On The Dopamine Transporter

The dopamine transporter (DAT) is an integral membrane protein that reuptakes dopamine (DA) from the extracellular space into the presynaptic neuron. DAT regulates dopaminergic neurotransmission as it maintains homeostatic synaptic DA levels in the brain. Psychostimulants such as cocaine disrupt DA homeostasis as it binds to DAT and prevents reuptake of DA. Excess DA in the synapse leads to prolonged dopaminergic neurotransmission, which is associated with cocaine related euphoria often leading to addiction. The DAT consists of 12 transmembrane domains (TMs) with N- and C-termini facing the cytoplasm. TMs 1, 3, 6, and 8 make up the core of the protein and the residues from these domains are involved in binding substrates and inhibitors. Although the effect of cocaine binding to DAT is known, the mechanism of DAT-cocaine interaction at the molecular level is still unknown. Therefore, to elucidate how cocaine binds to the DAT and how it is positioned in the binding pocket, we mapped the attachment site of the irreversible binding cocaine analogs, [125I]MFZ 2-24, [125I]RTI 82, and [125I]JHC 2-48. These compounds share a similar cocaine-based core structure, but have analog specific photo activatable side chains that extend from different regions of the cocaine core structure. Upon ultraviolet light activation, the photo activatable phenyl N3 (azido) group forms phenyl nitrene that becomes covalently attached to a residue on the protein, hence irreversible binding cocaine analogs. Previous studies narrowed the [125I]RTI 82 adduction site to the region surrounding TM6, between Ile291 and Arg344 on human DAT and between Met290 and Lys336 on rat DAT. The [125I]MFZ 2-24 attachment site was localized between residues Ile67 and Leu80 in TM1. To identify the specific amino acid attachment site of these analogs we created several methionine substitution mutants across TMs 1 and 6. This resulted in generation of custom cyanogen bromide (CNBr) cleavage sites. The results from peptide maps of photoaffinity labeled mutants proteolyzed with CNBr narrowed the adduction of [125I]MFZ 2-24 to Asp79 or Leu80 in TM1 and the adduction of [125I]RTI 82 to Phe320 in TM6. Trypsin and CNBr proteolysis of [125I]JHC 2-48 labeled rat DAT indicated a ligand attachment site C-terminal to TM6. Incorporation of three structural analogs to three distinct TM domains demonstrates that the appended azido groups on these analogs identify different faces of the ligand binding pocket. Thus, allowing for triangulation of cocaine orientation in its binding site via computational modeling

Understanding Addiction, Depression, And Autism Spectrum Disorder Through Structure-Function Analyses Of The Dopamine And Serotonin Transporters

The dopamine (DAT) and serotonin (SERT) transporters are monoamine neurotransmitter transporters (MATs) responsible for the reuptake of dopamine (DA) or serotonin (5-HT) from the synapse following vesicular release, effectively regulating synaptic neurotransmission. Blockade of these transporters by antagonists such as psychostimulant drugs or transporter mutations that affect function can compromise DA or 5-HT homeostasis and impact fundamental brain processes including movement, emotion, behavior, motivation, and memory. To address these issues, my dissertation research focused on: (1) identifying and characterizing the cocaine binding site in DAT, (2) determining the influence of membrane depolarization on DAT trafficking, (3) investigating antidepressant metabolite action in an antidepressant-insensitive mouse model, and (4) understanding the link between the structural and functional changes induced by mutations in SERT associated with autism spectrum disorder. Cocaine binding site in DAT – Through the development and utilization of several high affinity, photoactivatable cocaine ligands we identified a cocaine binding site in the core of DAT, a site that overlaps with the putative DA binding site, supporting a competitive mechanism for cocaine inhibition of DA uptake. (Chapters II-IV) Influence of membrane depolarization on DAT trafficking – Trafficking of mature DAT to and from the cell membrane is a highly regulated process. Within this process, we demonstrated that membrane depolarization alone could induce a CaMKIIα- and dynamin-dependent rapid reversible reduction in membrane DAT. (Chapter V) Characterization of antidepressant metabolites in the blockade of 5-HT reuptake – As studies of the antidepressant selective serotonin reuptake inhibitors (SSRIs) have revealed discrepancies between acute and chronic dosing treatments, we evaluated the sensitivity of SSRI metabolites in a mouse model of depression and identified a role for metabolites in antidepressant administration that may confound study conclusions. (Chapter VI) Autism-associated SERT mutations – Previously, several rare SERT coding variants were identified in humans with autism spectrum disorder that augment 5-HT transport function. We studied the structure of these variants and discovered alterations in SERT tertiary structure, which likely impact the catalytic activity or surface expression of SERT. (Chapter VII

Understanding The Structure-Function Relationships Between Monoamine Neurotransmitter Transporters And Their Cognate Ions And Ligands

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