Our Research

 

Dopaminergic System          

Projects in our laboratory are focused on the identification of novel mechanisms involved in regulation of dopamine neurotransmission in the brain. In particular we are interested in the regulation of dopamine transporter (DAT) an integral membrane protein vital for regulation of synaptic dopamine. Work in our laboratory is devoted to understanding the details of DAT regulation, trafficking, the pharmacological modulation of the transporter and how this is all integrated into the broader context of normal dopamine signaling and the pathogenesis of addiction, neurological and neuropsychiatric disorders.

 

Psychostimulants regulation of DAT activity

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca(2+)](i) from internal stores compared with AMPH. [Ca(2+)](i) responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca(2+)](i) and for the enhanced effects of METH on x[Ca(2+)](i) elevation. xCalmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca(2+) responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.

 

Partner proteins regulation of DAT function

1. α-synuclein regulation of dopamine transporter

Recent work in various disease models has begun to emphasize the significance of presynaptic dysfunction as an early event that occurs before manifestation of neurological disorders. An increasing number of proteins have been identified that interact with different domains of DAT and regulate the biology of the transporter. These interactions suggest that the synaptic distribution, targeting, compartmentalization, trafficking and functional properties of DAT can be regulated via interacting proteins. α-Synuclein is a 140-amino acid protein that forms a stable complex with DAT and is linked to the pathogenesis of neurodegenerative disease. To elucidate the potential functional consequences of DAT/α-synuclein interaction, we explored α-synuclein modulation of DAT activity in midbrain dopaminergic neurons obtained from TH::RFP mice, immortalized DA neurons, and a heterologous system expressing DAT. We used dual pipette whole cell patch clamp recording to measure the DAT-mediated current before and after dialysis of recombinant α-synuclein into immortalized DA neurons. Our data suggest that intracellular α-synuclein induces a Na+ independent but Cl--sensitive inward current in DAT-expressing cells. This current is blocked by DAT blocker GBR12935 and is absent when heat-inactivated α-synuclein is dialyzed into these cells. The functional consequence of this interaction on DAT activity was further examined with real-time monitoring of transport function using a fluorescent substrate of DAT, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+). Overexpression of α-synuclein in DAT-positive immortalized DA neurons and CHO cells expressing DAT decreased the magnitude and rate of DAT-mediated substrate uptake without a decrease in the initial binding of the substrate at the plasma membrane. Taken together our findings are consistent with the interpretation that DAT/α-synuclein interaction at the cell surface results in a DAT-dependent, Na+-insensitive, Cl-sensitive inward current with a decrease in substrate uptake, suggesting that DAT/α-synuclein interaction can modulate dopamine transmission and thus neuronal function.

 

2. Sigma-1R regulation of Dopamine transporter

Our recent findings suggest that Sigma-1R interacts with the dopamine transporter and methamphetamine exposure increases the frequency of this interaction.

 

Membrane localization of dopamine transporter

Though the trafficking of DAT to and away form the surface membrane has been studied extensively, it is unknown whether or not changes in the membrane potential also can regulate the trafficking of DAT. Our simultaneous patch-clamp and Total Internal Reflection Fluorescence (TIRF) confocal microscopy suggest that DAT changes in the membrane potential regulates the trafficking of Dopamine transporter. We found that membrane hyperpolarization increases YFP-DAT level at the membrane. The hyperpolarization-induced increase in surface DAT level is paralleled by an increase in DAT-mediated inward current. Currently we are examining the hypothesis that changes in the membrane potential regulate the trafficking of DAT to and from the cell surface membrane in the DA neurons.