Structure, Function and Pharmacology of Neurotransmitter Reuptake Systems

National Institute Of Mental Health

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Abstract

Drugs which act on plasma membrane neurotransmitter transport systems are among the most widely used treatments for neuropsychiatric disorders, including major depression and attention-deficit disorders. My laboratory has used cell biological, biochemical, physiological, and structural approaches to understand the function, regulation, and effects of drugs on neurotransmitter transporters. We have continued to explore the presynaptic actions of amphetamines by further examining the signaling events that they elicit within dopamine neurons. When amphetamine enters a dopamine neuron, it activates the small GTPases, RhoA and Rac1 and triggers internalization of the dopamine transporter (DAT) and a neuronal glutamate transporter (EAAT3) through a specialized RhoA-dependent internalization pathway. This removal of transporters from the cell surface results in the acute potentiation of both glutamate and dopamine signaling. We identified the trace amine receptor, TAAR1, as an intracellular GPCR that mediates the initial actions of AMPHs, trace amines, and other drugs, and showed that TAAR1 signals through two G-proteins, Gs and G13, to activate Protein Kinase A (PKA) and RhoA, respectively. In experiments on transgenic mouse lines lacking the TAAR1 receptor we have shown that the intracellular effects of amphetamine, including both the activation of PKA and RhoA, depend absolutely upon TAAR1. Using subcellularly-targeted FRET sensors we demonstrated that TAAR1-stimulated RhoA signaling occurs in a subcellular compartment on or adjacent to the endoplasmic reticulum, whereas PKA activation is distributed more broadly throughout the cell. Data using inhibitory G-protein alpha-subunit fragments suggest that there are two separate pools of TAAR1 receptor one poised to couple to RhoA and the other to cAMP signaling. We have continued to explore the role of TAAR1 as an intracellular target for amphetamines and shown that similar intracellular events occur in serotonin and norepinephrine neurons. Our recent work has shown that amphetamine also activates a trace amine receptor in norepinephrine neurons, and we have established that another amphetamine-related compound, MDMA, enters serotonin neurons through the serotonin transporter and activates closely related signaling cascades in serotonergic neurons. The ability of amphetamines to increase internalization of a cell surface glutamate transporter, EAAT3, and to potentiate excitatory inputs onto dopamine neurons was unexpected. Using a cell-permeant peptide that blocks EAAT3, but not DAT internalization we have been able to resolve the effects of amphetamine on excitatory neurotransmission in brain slices and in mice, using targeted viral expression. Surprisingly, these and additional studies that selectively delete the EAAT3 gene in dopamine neurons suggest that amphetamines effects on glutamate transporter trafficking determine the degree of locomotor activation observed following administration of the drug. We have also compared the effects of various amphetamine compounds on the activation of cellular signaling pathways. Comparison of the effects of methamphetamine to those of amphetamine indicate that while both treatments lead to a loss of cell-surface EAAT3, the effects of methamphetamine are observed in cells throughout the brain and do not depend on transport through DAT. These findings provide an explanation for the broader, more detrimental effects of methamphetamine: unlike amphetamine, methamphetamine has the capacity to alter EAAT3 surface expression and regulate excitatory neurotransmission, not only in dopamine neurons, but also in many other neuronal cell types within the brain.