Dysregulation of dopaminergic homeostasis has been established as the primary source of numerous neurological disorders including Parkinson’s and drug addiction. A tonic increase of dopamine (DA) in the nucleus accumbens is required for associating everyday events and behaviors with rewards. Yet many addictive exogenous compounds such as amphetamine (AMPH) and cocaine (COC) produce a much greater augmentation of synaptic DA levels that are linked to euphoria and a shift in behavior towards drug seeking. The protein responsible for maintaining extracellular levels of DA is the dopamine transporter (DAT). It is primarily located in the perisynaptic area at terminals of pre-synaptic neurons where its main function is to sequester DA from the extracellular space and to transport it back into the cell, a process that is electrogenic. AMPH and COC directly interact with DAT and alter its ionic currents. Not much is known about the effect of psychostimulant-induced DAT currents on neuronal excitability and neurotransmitter release. We use synthetic chemistry, molecular biology, and biophysics in heterologous expression systems to decipher the actions of drugs of abuse on DAT. Furthermore we demonstrate drug-induced DAT currents can activate Ca2+ channels associated with dopaminergic excitability. Lastly, we focused on investigating drug effects on excitability in a human midbrain dopaminergic cell line. Understanding how psychostimulants interact with DAT to produce the dysfunctional states of the transporter may facilitate the development of unique therapeutic strategies to treat psychostimulant dependence.

Research in drug development meets many challenges including lengthy, complex and costly procedures to identify novel pharmacotherapies. In our lab, we developed a method for fast screening of small molecules that interact with monoamine transports – dopamine and serotonin (DAT, SERT). These membrane proteins play important roles in brain neurotransmission responsible for cognition, motion and pleasure. Dysfunction in dopaminergic and serotonergic systems result in neurological disorders such as depression, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and addiction. DAT and SERT are responsible for uptake of dopamine (DA) or serotonin (5HT) into the synapse and they limit neurotransmitter signaling. Drugs that mimic or antagonize actions of endogenous neurotransmitters (DA and 5HT) increase the concentrations of DA and/or 5HT either by blocking the transporter (blockers) or by competing uptake with neurotransmitter (substrate). The uptake of substrates is associated to an inward current that depolarizes the cell membrane. Voltage-gated calcium channels (CaV) can respond to small changes in membrane potential. In our method, we combined permanent cell line expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) (FlpIn TREx expression system) with transient transfection of CaV. This system works as a tightly electrically coupled system. Cells challenged with substrate of the transports produce detectable Ca2+ signal while monoamine transporter blockers can inhibit these Ca2+ signals. The novelty of this method relies on the ability to discriminate between substrate and blockers of monoamine transporters. Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters. Our novel methodology can rapidly assess drug-effect profile on monoamine transporters and benefit development of new psychotherapeutics for treatment of mental illnesses. It can also be used to characterize mechanism of action of emerging drug of abuse, as well as to discover small molecules with novel drug-effect profile useful in basic neuroscience research.

Ligands of the human monoamine transporters encompass a wide range of both illicit and therapeutic drugs that act upon neural circuitry related to reward, motivation, and the processing of salient stimuli. The present study utilizes two methods for analyzing transporter substrates and inhibitors in order to characterize activity and assess potency. The first measures transient changes in intracellular calcium as a surrogate for transporter activity by harnessing the electrical coupling of monoamine transporters and L-type calcium channels. This is used to analyze novel chimera of the strong hDAT inhibitors methylphenidate and 𝛼-PPP in order to assess the contribution of specific moieties to potency. The observed reduction in potency suggests that methylphenidate may bind to the transporter in a manner distinct from 𝛼-PPP, as chimera would otherwise be expected to show similar activity to parent compounds. These results highlight the importance of 𝛼-carbon substituents and the relatively small contribution of beta-carbon groups to inhibitor potency at hDAT, while the lack of activity at hSERT suggests potency is not strongly influenced by beta-carbon or N-alkyl substituents. In order to further characterize drug-transporter interaction, a method was developed to analyze the kinetics of binding and unbinding using both known and novel hNET ligands, including a series of N-alkyl derivatives of 4-methylamphetamine. The study emphasizes the importance of both association and dissociation kinetics to affinity and sets up a methodological framework with two ways for determining Kd, with notable advantages over current models. The results indicate that lengthening the N-alkyl chain of 4-methylamphetamine leads to a decrease in potency and a shift in activity from substrate to blocker, with the results of N-propyl 4-methylamphetamine in particular indicating the potential existence of multiple low-affinity binding sites, each with distinct on and off kinetics. The implications of these results help elucidate the mechanism of action of transporter ligands and set up a framework for future studies that can more specifically classify the interaction between transporters and inhibitors or releasing agents.