Structure, Function and Pharmacology of Neurotransmitter Reuptake Systems

National Institute Of Mental Health

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Abstract

Our laboratory has shown that amphetamines trigger the internalization of the dopamine transporter (DAT) by a series of intracellular events that are distinct from the generally established actions of amphetamines to inhibit DA uptake or to increase DA efflux. We have found that when applied to cell lines, cultured DA neurons or midbrain slices, amphetamine activates the small GTPases, RhoA and Rac-1 and triggers internalization of the dopamine transporter (DAT) by a specialized internalization pathway that requires the activation of the small GTPase, RhoA. We also found that amphetamine must be transported into the cell to have these effects and its actions are blocked by cocaine, a drug that blocks DAT and prevents amphetamine entry. Elevation of cAMP, by DA receptors or by amphetamine-induced adenylate cyclase activation, inactivates RhoA and limits carrier internalization, consistent with roles for PKA- and Rho-dependent signaling in mediating the actions of amphetamines in dopamine neurons. Our work continues to explore our previous finding that a G-protein coupled trace amine receptor (TAAR1) serves as a direct intracellular target for amphetamines in dopaminergic, serotoninergic and noradrenergic neurons. Using transgenic mouse lines lacking the TAAR1 receptor we have shown that the intracellular effects of amphetamine, including both the elevation in cAMP and the increased RhoA activity, depend absolutely upon TAAR1 activation. We have shown that when activated by amphetamine within the cell, TAAR1 signals through a G-protein, known as G13 to activate RhoA and through another G-protein, Gs to increase cAMP. Using a series of subcellularly-targeted genetic sensors to detect RhoA or cAMP activation, we have been able to demonstrate that TAAR1 signaling initiates in an intracellular membrane compartment that is within or very proximal to the endoplasmic reticulum. We have also observed that the same amphetamine-activated RhoA-dependent mechanism downregulates a glutamate transporter, EAAT3, present on the surface of dopamine neurons. 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 vivo, 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 on glutamate transport activity to those of amphetamine indicate that while both treatments lead to a loss of cell-surface EAAT3, the effects of methamphetamine are much broader and do not depend on the expression of the 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. Our recent work has shown that amphetamine has similar actions on neurotransmission by norepinephrine by activating a trace amine receptor in norepinephrine neurons, and we have established that another amphetamine-related compound, MDMA, enters serotonin neurons through the 5-HT transporter (SERT) and activates similar G-protein coupled signaling cascades in serotonergic neurons.