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 dopamine (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. Intriguingly, amphetamine must be transported into the cell to have these effects and its actions are actually blocked by cocaine, a drug that inhibits DAT and prevents amphetamine entry. We have also found that elevation of cAMP, via DA receptors or by amphetamine-induced adenylate cyclase activation, inactivates RhoA and serves as a break on carrier internalization, thus demonstrating an important interaction between PKA- and RhoA-dependent signaling in mediating the actions of amphetamines. These observations also imply the existence of a novel intracellular target for amphetamines and suggest new cellular pathways to target in order to disrupt amphetamine action. In recent studies we have established that a G-protein coupled trace amine receptor (TAAR1) serves as a direct intracellular target for amphetamines in dopamine 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 are currently investigating where within the cell amphetamines activate TAAR1 and which G-proteins serve as downstream effectors. In other experiments we have shown that G-protein beta-gamma subunits released when G-protein-coupled receptors are activated bind directly to the DAT and enhance dopamine efflux. Using cell permeable peptide fragments and mutagenesis of the DAT we have been able to define the transporter domains required for this interaction and to develop structural models for how this interaction may facilitate dopamine efflux by the transporter. We have also observed that the same amphetamine-activated RhoA-dependent mechanism downregulates a glutamate transporter, EAAT3, present on the surface of dopamine neurons. We have identified the EAAT3 peptide sequence responsible for this regulation, generated a cell-permeant fusion protein that blocks internalization and have used it to explore the effects of amphetamine on excitatory neurotransmission in brain slices. These studies have provided new tools to distinguish the effects of amphetamine on dopaminergic and glutamatergic signaling. 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 suggest an explanation for the broader, more devastating 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. Glutamate transporters (also known as excitatory amino acid transporters or EAATs) present at the surface of neurons and supporting glial cells regulate the extracellular concentration of glutamate, the major excitatory neurotransmitter in the brain. By transporting glutamate back into the cell, these carrier proteins prevent glutamate from reaching toxic levels and also limit the extent and duration of transmitter signaling during glutamatergic neurotransmission. These carriers have an additional function in that they possess an anion channel activity that can regulate cellular excitability, which enables them to serve as sensors of glutamate levels outside the cell. Our laboratory has used site-directed mutagenesis, sulfhydryl modification, and chemical cross-linking approaches together with biochemical, and electrophysiological analyses of the mammalian carriers to examine the structural domains required for substrate transport and ion permeation. Although there has been progress towards understanding the mechanism of glutamate transport, little is known about the mechanism and structural basis of anion channel activation. We recently identified a conserved positively-charged arginine residue in transmembrane domain 7 as an essential element in the anion channel gating mechanism. Substitution of this residue with a negatively-charged amino acid, eliminates sodium- and substrate-dependent anion channel gating, and drives the channel into a substrate-independent constitutively open state. Our data suggest that anion channel gating occurs through a transition from intermediate conformations that are closely linked to transport. We hypothesize that interactions of this crucial cationic residue with nearby anionic groups are required to stabilize the closed state of channel and thus are intrinsic elements of the channel gating mechanism. We created a homology model of the transporter and have identified potential interacting residues that also appear to be required for anion channel gating in EAATs. The neuronal glutamate transporter isoform, EAAT3 has also been shown to transport the amino acid cysteine and has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a molecule critical for preventing oxidative stress and neuronal toxicity. This year the laboratory also completed work examining the mechanism of cysteine transport by EAAT3. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.

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3
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2015
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U.S. National Institute of Mental Health
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Underhill, Suzanne M; Ingram, Susan L; Ahmari, Susanne E et al. (2018) Neuronal excitatory amino acid transporter EAAT3: Emerging functions in health and disease. Neurochem Int :
Lohani, Sweyta; Martig, Adria K; Underhill, Suzanne M et al. (2018) Burst activation of dopamine neurons produces prolonged post-burst availability of actively released dopamine. Neuropsychopharmacology 43:2083-2092
Cheng, Mary Hongying; Garcia-Olivares, Jennie; Wasserman, Steven et al. (2017) Allosteric modulation of human dopamine transporter activity under conditions promoting its dimerization. J Biol Chem 292:12471-12482
Li, Ming-Hua; Underhill, Suzanne M; Reed, Cheryl et al. (2017) Amphetamine and Methamphetamine Increase NMDAR-GluN2B Synaptic Currents in Midbrain Dopamine Neurons. Neuropsychopharmacology 42:1539-1547
Insel, Paul A; Amara, Susan G; Blaschke, Terrence F et al. (2017) Introduction to the Theme ""New Methods and Novel Therapeutic Approaches in Pharmacology and Toxicology"". Annu Rev Pharmacol Toxicol 57:13-17
Cheng, Mary Hongying; Torres-Salazar, Delany; Gonzalez-Suarez, Aneysis D et al. (2017) Substrate transport and anion permeation proceed through distinct pathways in glutamate transporters. Elife 6:
Garcia-Olivares, J; Baust, T; Harris, S et al. (2017) G?? subunit activation promotes dopamine efflux through the dopamine transporter. Mol Psychiatry 22:1673-1679
Hong, Weimin Conrad; Yano, Hideaki; Hiranita, Takato et al. (2017) The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats. J Biol Chem 292:11250-11261
Divito, Christopher B; Borowski, Jenna E; Glasgow, Nathan G et al. (2017) Glial and Neuronal Glutamate Transporters Differ in the Na+ Requirements for Activation of the Substrate-Independent Anion Conductance. Front Mol Neurosci 10:150
Mortensen, Ole V; Larsen, Mads B; Amara, Susan G (2017) MAP Kinase Phosphatase 3 (MKP3) Preserves Norepinephrine Transporter Activity by Modulating ERK1/2 Kinase-Mediated Gene Expression. Front Cell Neurosci 11:253

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