The dopamine transporter (DAT) is responsible for actively regulating the concentration of extraneuronal dopamine in the central nervous system. As a member of the neurotransmitter/sodium symporter (NSS) family of membrane proteins, the DAT serves to shuttle extracellular dopamine molecules and Na+ ions back across the lipid bilayer, harnessing electrochemical potential energy inherent to the cellular transmembrane Na+ gradient to catalyze the thermodynamically unfavorable process of moving dopamine back against its concentration gradient. Dopaminergic signaling plays a critical role in cognition, motor function, affect, motivation, neuroeconomics and behavioral reinforcement and DAT function is implicated in a number of neuropsychiatric disorders (e.g. attention deficit disorders (ADD/ADHD), parkinsonian disorders, anhedonia/depression, compulsive behavior and addiction). The DAT is also of significant pharmacological interest—it is the target of various clinically used psychostimulant, nootropic and antidepressant compounds, such as dextroamphetamine, modafinil, methylphenidate and bupropion.
In addition, the DAT is the principle target of perhaps the most addictive drug known to man: cocaine, which is used as a recreational stimulant drug and is consumed by humans via intranasal or intravenous administration of (-)-cocaine HCl powder, or alternatively, by vaporization of the freebase alkaloid (‘crack’). One particularly enigmatic aspect of DAT pharmacology is the disparate reinforcing efficacy (‘addictiveness’) of various DAT ligands—a given DAT-affecting molecule may have dramatic, mild or even a complete lack of behaviorally rewarding effects, regardless of absolute binding affinity. For example, the atypical DAT inhibitors benztropine and GBR12909 do not share cocaine’s notorious addictive liability, despite having greater affinity for the transporter. DAT ligands with differing chemical structures exert unique conformational effects on the transporter protein; the nature of these conformational effects determines a ligand’s reinforcing efficacy. In this sense, the DAT appears to behave like a classically defined receptor: chemically distinctive ligands exert specific conformational effects upon formation of a DAT/ligand complex and these conformational changes are transduced via cellular signaling cascades, ultimately eliciting unique behavioral and psychical effects.
The overarching goal of my thesis research has been to investigate and further define these receptor-like properties. DAT mutants with a biased conformational equilibrium have been useful in highlighting different actions of cocaine-like and atypical DAT ligands. In this thesis, I employ two point-mutated DATs (W84L and D313N) that exhibit a conformational bias towards the outward-facing open state to investigate the differential binding mechanisms of four classes of DAT ligands: cocaine-like inhibitors, atypical inhibitors, substrates-like molecules and a novel class of bivalent substrate-like DAT inhibitors. By increasing the likelihood that the DAT will adopt an outward-facing conformational state, these mutations increase the affinity of cocaine-like inhibitors considerably, but have little or opposite effect on atypical inhibitor binding. Whereas cocaine and structural analogues preferentially stabilize the DAT in an outward-facing conformation, atypical inhibitors (lacking cocaine-like potential for addiction), such as benztropine, GBR12909 and modafinil stabilize a more closed inward-facing or occluded conformation. Using pharmacological probes, conformation-affecting assay conditions and in silico computational modeling, I show that the transporter-binding mechanism of atypical DAT inhibitors—including structural analogues of GBR12909 and benztropine, modafinil and bupropion—is distinct from that of cocaine and other phenyltropanes (e.g. β-CFT). This idea is in fact consistent with the preclinical literature, which suggests that substrates (dextroamphetamine) and atypical DAT inhibitors (modafinil and the benztropines) are more effective as treatments for cocaine addiction than methylphenidate, which preferentially interacts with the same transporter conformation as cocaine. Notably, I also show that (at least for the ligands investigated here) the presence of a diphenylmethoxyl moiety was sufficient (but not necessary) to engender a given DAT inhibitor molecule with an atypical binding profile. Overall, the results highlight a mechanistic difference between atypical and cocaine-like stimulants and further demonstrate that the conformational effects of a given DAT inhibitor influence its phenomenological effects.
My findings also demonstrate that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors, until a critical distance threshold is reached. The most potent bivalent compound (bearing two dopamine-like pharmacophoric ‘heads’ separated by an 8-carbon linker) achieved an 82-fold gain in inhibition of [3H]CFT binding compared to dopamine itself and bivalent compounds with a 6-carbon linker and heterologous combinations of dopamine-, amphetamine- and β-phenethylamine heads all resulted in considerable gains in DAT affinity. In silico modeling of the DAT protein (based upon crystals of the prokaryotic neurotransmitter symporter homologue LeuTAa) and docking of the most potent bivalent ligands suggested simultaneous occupancy of two discrete substrate-binding domains. These studies represent the first assessment of bivalent substrate-like DAT ligands and the first presentation of both pharmacological and computational evidence indicating the existence of multiple substrate-interaction sites in a single DAT protein. The findings also imply that it is possible to design novel types of DAT inhibitors based upon the “multivalent ligand” strategy.
Finally, I conclude with preliminary experiments designed to determine how various DAT ligands—acting either as amphetaminergic substrates, cocaine-like nontranslocated inhibitors or atypical inhibitors—affect transporter trafficking and oligomerization state. The original notion that monoamine transporters are static ‘molecular pumps’ has changed dramatically over the last decade, with contemporary studies indicating that NSS proteins are highly dynamic entities that rapidly cycle between the plasma membrane and endosomal compartments. Moreover, there is robust evidence to suggest that NSS proteins exist as oligomers at the plasma membrane. I show results of initial surface biotinylation and confocal imaging experiments investigating the trafficking effects of different classes of DAT ligands and also present pilot data from the Förster resonance energy transfer (FRET) microscopy protocol developed during the final portion of my thesis project.
|Advisor:||Reith, Maarten EA|
|Commitee:||Carr, Kenneth, Rice, Margaret, Rothman, Richard, Simon, Eric|
|School:||New York University|
|Department:||Basic Medical Science|
|School Location:||United States -- New York|
|Source:||DAI-B 73/03, Dissertation Abstracts International|
|Subjects:||Molecular biology, Pharmacology, Biochemistry|
|Keywords:||Bivalent ligands, Cocaine addiction, Dat mutants, Differential interaction, Dopamine transporter (dat), Nss proteins, Phenethylamines|
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