Academic literature on the topic 'Drosophila melanogaster dopamine transporter'

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl− binding on [3H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (Kd) for [3H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.

The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds.

Non-enzymatic glycation and covalent modification of proteins leads to Advanced Glycation End products (AGEs). AGEs are biomarkers of aging and neurodegenerative disease, and can be induced by impaired neuronal signaling. The objective of this study was to investigate if manipulation of dopamine (DA) in vitro using the model protein, bovine serum albumin (BSA), and in vivo using the model organism Drosophila melanogaster, influences fluorescent AGEs (fAGEs) formation as an indicator of dopamine-induced oxidation events. DA inhibited fAGEs-BSA synthesis in vitro, suggesting an anti-oxidative effect, which was not observed when flies were fed DA. Feeding flies cocaine and methamphetamine led to increased fAGEs formation. Mutants lacking the dopaminergic transporter or the D1-type showed further elevation of fAGEs accumulation, indicating that the long-term perturbation in DA function leads to higher production of fAGEs. To confirm that DA has oxidative properties in vivo, we fed flies antioxidant quercetin (QUE) together with methamphetamine. QUE significantly decreased methamphetamine-induced fAGEs formation suggesting that the perturbation of DA function in vivo leads to increased oxidation. These findings present arguments for the use of fAGEs as a biomarker of DA-associated neurodegenerative changes and for assessment of antioxidant interventions such as QUE treatment.

Dopamine is a neuromodulator that is secreted to the synapse to relay chemical signals to target neurons. Abnormal levels of dopamine release leads to various neurodegenerative diseases. Therefore, measuring dopamine is essential to understand how dopamine is regulated under normal and pathological conditions. Drosophila melanogaster, the fruit fly, is an ideal model system for studying fundamental neurological processes and diseases because of the availability of sophisticated genetic tools and well conserved neurological processes between mammals and flies. Majority of neuroscience studies involved in modifying a gene and measure the effect of genetic mutation on output behaviors. However, dopamine release is highly dynamic because of the complex activity of dopamine transporters and autoreceptors. Therefore, to understand how dopamine signaling controls the behavior, a direct measurement of changes in dopamine release is necessary. Fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode is an electrochemical technique that trace concentration changes in dopamine release on the sub-second time scale. Our lab pioneered directly measuring various endogenous neuromodulators in the fly central nervous system with FSCV. Initially, these studies were performed in ex vivo preparations, where brains were isolated from larvae and adult fly, and thus could not monitor neuromodulators during behavior. In this study, we developed in vivo FSCV method to measure phasic dopamine in the mushroom body (MB) during behavior for the first time. The MB in fly has been extensively studied as an associative center for regulating olfactory learning and memory. First, acetylcholine stimulation was applied to the MB heel and medial tip to characterize dopamine signaling and to demonstrate the feasibility of in vivo FSCV in intact fly brain. Application of 0.2 pmol acetylcholine released 0.36 ± 0.06 µM dopamine in the medial tip, which is slightly higher than 0.22 ± 0.06 µM dopamine in the heel. Compartmental differences in evoked release suggest heterogeneity of dopamine regulation in the MB. Nisoxetine, a dopamine transporter inhibitor, and flupentixol, a D2 antagonist, increased stimulated dopamine release. We then applied the in vivo method to monitor changes in behaviorally evoked dopamine release during sugar feeding. Sugar feeding evoked 0.31 ± 0.09 µM dopamine in the medial tip of MB. Flupentixol significantly increased sugar evoked release implying D2 receptor acts as autoreceptor and regulates dopamine signaling during sugar feeding. Therefore, this developed in vivo FSCV method is a great addition to the existing tools to measure endogenous neuromodulators in the fly and valuable for studying real-time dopamine signaling during behavior. This in vivo method also can be further extended to better understand how dopamine and other neuromodulators regulate complex behaviors, such as reward associated learning and memory formation.

Drosophila’s white gene encodes an ATP-binding cassette G-subfamily (ABCG) half-transporter. White is closely related to mammalian ABCG family members that function in cholesterol efflux. Mutants of white have several behavioral phenotypes that are independent of visual defects. This study characterizes a novel defect of white mutants in the acquisition of olfactory memory using the aversive olfactory conditioning paradigm. The w1118 mutants learned slower than wildtype controls, yet with additional training, they reached wildtype levels of performance. The w1118 learning phenotype is also found in the wapricot and wcoral alleles, is dominant, and is rescued by genomic white and mini-white transgenes. Reducing dietary cholesterol strongly impaired olfactory learning for wildtype controls, while w1118 mutants were resistant to this deficit. The w1118 mutants displayed higher levels of cholesterol and cholesterol esters than wildtype under this low-cholesterol diet. Increasing levels of serotonin, dopamine, or both in the white mutants significantly improved w1118 learning. However, serotonin levels were not lower in the heads of the w1118 mutants than in wildtype controls. There were also no significant differences found in synapse numbers within the w1118 brain. We propose that the w1118 learning defect may be due to inefficient biogenic amine signaling brought about by altered cholesterol homeostasis.