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. 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 have shown that the 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. In another series of 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 in cultured cells and in vivo. 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 DA efflux by the transporter. Our work has demonstrated that release of G-beta-gamma subunits triggered by endogenous receptor activation is sufficient to enhance DA efflux in cultured cells and neurons. Most recently we have shown that the G-beta-gamma subunit drives the transporter into a novel conformation that facilitates dopamine efflux through a novel pathway that appears distinct from the pathway for transport. 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. Recent work has been directed at understanding the mechanism and structural basis of anion channel activation. We have constructed a computational model of a glutamate transporter isoform, EAAT4, which includes the cytoplasmic C-terminus. Our model suggests potential interactions between charged residues in the C-terminus and in a conserved region of transmembrane domain 3 (TM3) that may be relevant to function. We first tested this hypothesis using electrophysiological recordings in Xenopus oocytes expressing different point mutations in the two domains and using C-terminal truncations. Our preliminary results demonstrated that truncation of the full C-terminus disrupts channel gating, drives the channel into an open state and significantly reduces glutamate transport, suggesting that the C-terminal domain and its potential interaction with TM3 may be critical for the structural coupling between substrate transport and anion channel opening. To further validate the possibility that the C-terminal-TM3 interaction regulates the coupling between substrate transport and channel gating, we designed cell-permeable peptides that correspond to both regions and applied different concentrations of either peptide to oocytes expressing the full-length WT EAAT4. Our hypothesis was that these peptides would compete with and disrupt the C-terminal-TM3 interaction in a manner similar to the C-terminal truncation. Indeed, application of either peptide to EAAT4-exressing oocytes yielded results identical to the phenotype observed with the truncation mutants: glutamate-independent currents consistent with an open channel state and a reduced transport activity. These interfering peptides now provide new tools to address the structural basis for the coupling between anion channel opening and substrate translocation.

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7
Fiscal Year
2019
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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
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
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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
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

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