Type of Document Dissertation Author Solis, Ernesto URN etd-11302012-133021 Title Monoamine Transporter Substrates and Inhibitors Degree PhD Department Neuroscience Advisory Committee
Advisor Name Title Kevin P. Currie Committee Chair Louis J. DeFelice Committee Chair Maureen K. Hahn Committee Member Randy D. Blakely Committee Member Sandra J. Rosenthal Committee Member Keywords
- fluorescent substrates
- serotonin transporter
- dopamine transporter
Date of Defense 2012-09-28 Availability unrestricted AbstractA myriad of human behaviors, such as mood, awareness and motivation, are modulated by the monoamine neurotransmitters serotonin, norepinephrine and dopamine, respectively. Consequently, dysfunction of these monoaminergic systems underlies numerous medical conditions. In particular, disturbances in the serotonergic system are implicated in depression, bipolar disorder, and autism, whereas the dopaminergic system is implicated in Parkinsonâ€™s disease and addiction. During neurotransmission high concentrations of monoamine neurotransmitters are released from presynaptic neurons into the synaptic cleft where they diffuse to bind and activate pre- and postsynaptic receptors. The primary way to terminate neurotransmission involves monoamine transporters, which shuttle monoamines back into presynaptic neurons where they replenish synaptic vesicle contents. The monoamine transporters are molecular targets for antidepressants and psychostimulants that function to increase monoamine levels in the brain. For example, serotonin transporter (SERT) reuptake is inhibited by Prozac to increase serotonin levels and treat various mood disorders. Similarly, dopamine transporter (DAT) reuptake is altered with drugs, such as cocaine or amphetamine, which results in enhanced dopaminergic signaling and is thought to underlie reward and addictive behaviors. Transport through the monoamine transporters is not thoroughly understood, and the traditional model with fixed substrate-ion stoichiometry has been challenged in recent years with the discovery of ionic currents mediated by monoamine transporters. In an effort to better understand the activity of monoamine transporters, a variety of substrates and inhibitors are utilized. In particular, in my work I characterize fluorescent compounds that are based on a known monoamine transporter substrate and describe their utility as reporters to study serotonin transporter activity in real-time. In addition, I describe a novel effect induced by amphetamine and related compounds at both DAT and SERT whereby even after external removal of these compounds, a persistent current remains. These studies provide information about various substrates that exert an array of distinct effects on SERT and DAT, which may enable further studies to elucidate the nature of transporter biophysics.
1. APP+ is a Fluorescent Substrate for the Serotonin Transporter
One limitation to transporter research is the inability to monitor substrate uptake in real-time. Traditional methods such as radiolabeled uptake assays, though highly specific, yield poor temporal resolution. Electrophysiology on the other hand provides excellent time resolution, but currents are mediated mostly by ionic fluxes and therefore do not yield direct information about substrate transport. To investigate this issue, we collaborated with Dr. Ian D. Tomlinson and the laboratory of Dr. Sandra Rosenthal to develop compounds based on a known monoamine transporter substrate. We identified and characterized a fluorescent compound called APP+ that is suitable to monitor SERT transport in real-time. We employed a range of techniques to elucidate thoroughly the specificity of this compound for SERT expressed in Xenopus laevis oocytes and mammalian cells. Finally, we used APP+ to study binding and transport through SERT. This work will help to uncover fundamental information about hSERT, and to improve our ability to study these transporters.
2. Amphetamine Induces a Persistent Leak Current in the Dopamine Transporter
Amphetamine (AMPH) and related compounds increase dopamine (DA) levels in the brain and cause profound behavioral effects. One target for these drugs is the dopamine transporter (DAT) that normally regulates synaptic DA levels. DAT agonists, such as DA and AMPH, induce DAT-mediated currents driven by sodium. By measuring DAT currents on voltage-clamped Xenopus laevis oocytes, we discovered a DAT leak current induced by external exposure to the S(+)amphetamine (S(+)AMPH) enantiomer that persists long after its removal. We determined that the AMPH-induced leak current in DAT depends on sodium and is blocked by cocaine. In addition, intracellular application of S(+)AMPH can induce the leak current effectively, which suggests an internal secondary binding site in DAT. Understanding this novel effect of amphetamine on DAT has implications in the understanding of human behavior because amphetamine-induced persistent currents likely impact dopaminergic signaling, DA release mechanisms, and amphetamine abuse.
3. A Comparison of Leak and Persistent Leak Currents Induced by Methamphetamine and 3,4-Methylenedioxymethamphetamine in the Human Dopamine and Serotonin Transporters
After establishing the S(+)amphetamine-induced persistent leak current at DAT, we expanded this work to test if other DAT-mediated releaser agents related to AMPH would also induce the persistent leak current. In particular, we focused on 3,4-methylenedioxy-methamphetamine (MDMA) and methamphetamine (METH) because it is known that although MDMA and METH are structurally related they exert distinct behavioral effects in people. However, since MDMA has preference for the serotonin transporter and METH acts more potently at the dopamine transporter, we made a comparison of the effects of METH and MDMA on both SERT and DAT. Lastly, we uncovered that the amphetamine derivative para-chloroamphetamine (pCA) confers a substantial persistent leak current at the human serotonin transporter. These findings could open new avenues towards the study of the effect drugs of abuse have on behavior.
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