Regulation of Transporter Function and Trafficking by Amphetamines
Amphetamine increases extracellular dopamine through several mechanisms including internalization of the dopamine transporter, DAT. Recently, we further characterized the mechanism of endocytosis as clathrin-independent, dynamin-dependent and mediated by activation of the small GTPase RhoA. Modulation of this pathway leads to behavioral modification of amphetamine responses in vivo. We are currently working on identifying the intracellular target for amphetamine that initiates this cascade.
Amphetamine also modulates extracellular glutamate concentrations and we discovered that the excitatory amino acid transporter EAAT3 is also internalized by amphetamine. This response leads to potentiation of glutamatergic neurotransmission in dopamine neurons. By mutational analysis, we found a unique amino acid sequence on EAAT3 that mediates this response. Using this and other tools, we are currently characterizing the mechanism of EAAT3 internalization, identifying precisely where this occurs, how long it persists and the behavioral consequences of EAAT3 internalization.
Using microscopy, biochemical assays and behavioral evaluation we are further characterizing amphetamine-stimulated neurotransmitter transporter trafficking as well as identifying other compounds that modulate surface localization of monoamine and glutamate transporters.
Tools and Techniques
We use a variety of in vitro biochemical techniques, cell-line and primary culture models as well as acute brain slice preparations and in vivo assays. Analysis is carried out with biochemical assays, microscopy (confocal, TIRF, FRET, SIM/STORM) and behavioral assays.
Suzanne Underhill PhD (principal)
Jingshan Chen PhD
Wheeler DS, Underhill SM, Stolz DB, Murdoch GH, Thiels E, Romero G Amara SG (2015) Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine. Proc Natl Acad Sci USA. 112(51): E7138-47. PMID: 26553986
Underhill SM, Wheeler DS, Li M, Watts SD, Ingram SL, Amara SG (2014) Amphetamine modulates excitatory neurotransmission through endocytosis of the glutamate transporter EAAT3 in dopamine neurons. Neuron; 83(2):404-16. Doi: 10.1016/j.neuron.2014.05.043. PMID: 25033183 PMCID: PMC4159050
Structure-function Relationships in Excitatory Amino Acid Transporters (EAATs)
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Extracellular glutamate concentrations are highly regulated by the members of the Excitatory Amino Acid Transporter (EAAT) family. This family of integral plasma membrane proteins possesses two distinct functional activities: an ion-coupled co-transport process that moves glutamate into the cell, which maintains low levels of extracellular glutamate in the synaptic space; and a substrate-gated anion channel, that regulates cellular excitability.
Although the molecular basis of substrate transport is not fully understood, our understanding of this mechanism has been significantly expanded in the last 15 years by a number of functional and crystallographic studies, revealing critical features of the domains and residues required for substrate recognition, binding and translocation. On the contrary, the mechanism and structural features underlying anion channel function remain elusive. In our laboratory, we aim to elucidate the structural components controlling anion channel permeation trough EAATs, as well as the structural coupling between the two transport mechanisms.
To do that we combine molecular biology, radiolabeled glutamate uptake, cysteine modification experiments and electrophysiological recordings in heterologous expression systems expressing the different members of the EAAT family.
We have two ongoing collaborations with two computational labs. With Dr. Jeff Comer at Kansas State University, we are continuing our investigation to understand the molecular determinants responsible for anion channel gating in EAATs. With Dr. Ivet Bahar at the University of Pittsburgh we are condicting Molecular Dynamic simulations to identify the residues lining the anion permeation pathway also in EAATs.
Delany Torres-Salazar PhD (principal)
Jennie Garcia-Olivares PhD
Torres-Salazar D, Gonzalez-Suarez AD, Amara SG (2016) Transport and channel functions in EAATs: the missing link. Channels (Austin). 10(2):86-7. PMID: 26683197
Torres-Salazar D, Jiang J, Divito CB, Garcia-Olivares J and Amara SG (2015) A Mutation in Transmembrane Domain 7 (TM7) of Excitatory Amino Acid Transporters Disrupts the Substrate-dependent Gating of the Intrinsic Anion Conductance and Drives the Channel into a Constitutively Open State. J Biol Chem. 290 (38): 22977-90. PMID: 26203187
Dopamine Transporters (DAT) Modulation by GPCRs
Proper function of reward circuitry within the brain requires that the presynaptic dopamine transporter (SLC6A3, DAT) efficiently recaptures dopamine (DA). DAT function can be regulated by many intracellular mechanisms including phosphorylation, ubiquitination, and protein-protein interactions. We recently reported a novel mechanism describing the regulation of DAT by heterotrimeric G-proteins (1).
We found that Gβγ subunits bind directly to the C-terminus of DAT (residues 582-620), and upon G-protein activation, the release of Gβγ results in a decrease in DA uptake. In our group, with the collaboration of Dr Gonzalo Torres, at the University of Pittsburgh, we explore the molecular basis of this interaction. To further understand the mechanisms of DAT regulation by Gβγ subunits we use different approaches, protein biochemistry, binding assays and functional assays, to analyze the molecular basis of the protein-protein interaction and their functional consequences.
These mechanisms might be relevant not only to the action of psychostimulants – and thus, relevant to drug addiction – but also to physiological mechanisms by which dopamine neurons release dopamine through the transporter. These kinds of hypotheses can be tested in heterologous systems, additionally; the use of genetically modified animal models would expand the results to in vivo approaches and bring and provide an approximation to a model that could explain clinical aspects of drug addiction and mood disorders.
Jennie Garcia-Olivares PhD (principal)
Delany Torres-Salazar PhD
Dr. Abigail Nash
Garcia-Olivares J, Torres-Salazar D, Owens WA, Baust T, Siderovski DP, Amara SG, Zhu J, Daws LC, Torres GE (2013) Inhibition of Dopamine transporters activity by G protein Bγ subunits PLoS One; 8(3):e59788. PMID: 23555781
Genetic and Functional Analyses of Human Trace Amine Receptors
We are interested in molecular mechanisms of pathogenesis of psychiatric disorders with abnormal dopamine neurotransmission. Our recent research focuses on the roles of human trace amine receptor 1 (TAAR1) in dopamine neurotransmission and functional single nucleotide polymorphisms (SNPs) in the TAAR1 gene. TAAR1 is a unique G-protein coupled receptor that can be activated by trace amines, amphetamine and dopamine.
However, its role in dopamine neurotransmission remains unknown or controversial. We are applying inducible transgenic technology and knockout technologies including CRISPR and TALEN to the development of genetic cellular and animal models carrying human mutations, especially functional SNP mutations within the TAAR1 gene. We have recently identified functional SNPs in human TAAR1 gene and developed anti-TAAR1 CRISPRs for knocking out TAAR1 gene in human cell lines including induced pluripotent stem cell (iPSC) lines. The genetic cellular models will be used for testing the effect of the functional SNP mutations on dopamine neurotransmission.
These studies could provide insight into the molecular mechanisms of abnormal dopamine neurotransmission in psychiatric disorders, generate new cellular and animal models for testing novel drugs that target TAAR1, and validate functional SNPs for future predictive and individualized medicine.
Jingshan Chen PhD (principal)