Gotowa bibliografia na temat „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.

Drosophila melanogaster has a long history of being used as an animal model for a wide variety of human diseases, including genetic diseases, neurodegeneration and alcoholism. Despite the fact that Drosophila was first used as an animal model over 100 years ago, it still remains an extremely relevant model today, thanks to its short life cycle, its low cost ease to rear in laboratory conditions and the sophistication of the molecular tools available for genetic manipulation in Drosophila melanogaster. This model also has far less genetic redundancy with respect to mammals, making the study of the role of certain genes far more straightforward, and yet despite this, still possesses an ortholog for 75% of human disease-causing genes. All of these properties contribute to the relevance of this model and were taken advantage of during this project. In the first part of this project, Drosophila melanogaster was used as a model for mitochondrial deoxynucleotide transport. The Drosophila homolog CG18317 of the yeast gene RIM2, which was previously reported to be a pyrimidine dNTP transporter, was characterized. Knock-out (K.O.) flies for gene CG18317, here referred to as drim2, were characterized for mitochondrial function and mtDNA integrity. The two human homologs for this gene, PNC-1 and SLC25A36 were also expressed in this mutant background, in order to investigate the functional homology of these genes and confirm the validity of this model for human mitochondrial dNTP transport. This project also focuses on further characterizing a K.O. fly line for dTTC19, a gene whose human homolog has already been tied to mitochondrial encephalopathy and psychosis in humans. This characterization was also accompanied by the generation of three K.O. lines which express the dTTC19 gene in a mutant background, in order to finally confirm that the entirety of the mutant phenotype is due to the absence of transcription of the dTTC19 gene. Finally, this project attempts to propose a new protocol which will enable researchers to use Drosophila melanogaster as a model for neurological disorders which present with antisocial symptoms. A protocol was developed to investigate social behaviour in Drosophila melanogaster and to demonstrate that subtle changes in either dopamine levels or previous social contact can have dramatic effects on their social interactions. We therefore propose that Drosophila can also be a useful model for the investigation of the genetic factors involved in diseases which present with antisocial behaviour such as autism, obsessive compulsive disorder, depression and so forth. In conclusion, this project takes full advantage of Drosophila melanogaster as an animal model for mitochondrial biology and disease. Furthermore, it proposes yet another way in which Drosophila can be used as a model which has not yet been done.
Drosophila melanogaster ha una lunga storia come animale modello per tante malattie umane, incluse le malattie genetiche, la neurodegenerazione e l’alcolismo. Anche se Drosophila fu inizialmente utilizzata come animale modello più di 100 anni fa, rimane comunque un modello rilevante oggi grazie al suo ciclo vitale breve, il suo basso costo e la sofisticazione degli attrezzi molecolari disponibili per la sua manipolazione genetica. Questo modello ha anche meno ridondanza genetica rispetto ai mammiferi, rendendo lo studio della funzione di questi geni molto più diretto, ma malgrado questo possiede un ortologo per 75% dei geni legati a malattie umane. Tutte queste proprietà contribuiscono alla sua rilevanza come modello e sono state sfruttate durante questo progetto. In primis, Drosophila melanogaster è stata usata come modello per il trasporto mitocondriale di deossinucleotidi. Il gene RIM2 in lievito, che è stato precedentemente caratterizzato come trasportatore mitocondriale di deossinucleotidi pirimidinici, ha un omologo in Drosophila: CG18317, qui chiamato drim2, che è stato caratterizzato in questo progetto. Questo gene è stato rimosso in vivo e la funzione mitocondriale e l’integrità del mtDNA sono state caratterizzate. I due omologhi umani per questo gene, PNC-1 e SLC25A36, sono stati espressi nel mutante, per determinare l’omologia funzionale di questi geni e per confermare la validità di questo mutante come modello per il trasporto mitocondriale umano di deossinucleotidi. Questo progetto si è anche focalizzato su una caratterizzazione più approfondita di una linea mutante per dTTC19, un omologo di un gene umano che è già stato collegato alla encefalopatia mitocondriale e la psicosi. Questa caratterizzazione è stata accompagnata dalla generazione di tre linee mutanti che esprimono dTTC19, per confermare che il fenotipo mutante osservato sia dovuto alla mancanta trascrizione di dTTC19. In fine, questo progetto propone un nuovo protocollo che, nella nostra opinione, permetterà di utilizzare Drosophila melanogaster come modello per disturbi neurologici che presentano con sintomi asociali. Un protocollo è stato sviluppato per studiare il comportamento sociale in Drosophila melanogaster e per dimostrare che piccole differenze nei livelli di dopamina o nel contatto sociale dopo l’eclosione possono avere effetti drammatici sulle interazioni sociali in Drosophila. Proponiamo che Drosophila può essere un modello utile per lo studio dei fattori genici coinvolti nelle malattie che presentano con comportamento asociale come l’autismo, il disturbo ossessivo compulsivo, la depressione eccetera. In conclusione, questo progetto sfrutta interamente Drosophila melanogaster come animale modello per la biologia e le malattie mitocondriali. In più, propone un nuovo modo per utilizzare Drosophila come modello che non è stato finora sfruttato.

Le comportement sexuel des drosophiles est déterminé génétiquement, il fait intervenir différents mécanismes de communication (chimique: phéromones, acoustique: chant et visuel) et des comportements plus simples (activité locomotrice). Il a de plus été démontré que les enzymes finales de la synthèse de la dopamine (DDC: dopa décarboxylas et TH:tyrosine hydroxylase) étaient produites sous la cuticule et dans le SNC. Dans un premier temps, la recherche s'est portée sur d'éventuelles différences des hydrocarbures cuticulaires dues à la présence ou à l'absence de dopamine. Pour cela on utilise des approches pharmacologiques et génétiques: des inhibiteurs d'enzymes de la TH sur des souches sauvages, des souches mutantes (Ddc ts) complémentées avec les composés qui leur font défaut et des souches mutantes au sein des gènes de structure: Ddc et pale. On observe alors une diminution corrélée de la quantité de dopamine et des hydrocarbures femelles spécifiques. Par la suite, nous avons montré que ce défaut, comparable à l'effet de la décapitation est compensé par une application topicale unique chez ces femelles. Dans un deuxième temps, différents aspects du comportement ont été analysé. Nous avons montré que les mouches mâles ayant une quantité de dopamine et de sérotonine réduite ont une plus faible réussite à la copulation, une plus faible activité locomotrice et un chant modifié. La dopamine semble donc impliquée dans la régulation du comportement sexuel chez la drosophile à différents niveau: production phéromonale, production acoustique et activité locomotrice. Enfin, une étude des variations de production phéromonale en fonction de la température réalisée sur deux souches sauvages de type Drosophila melanogaster permet de conclure à une augmentation de la production des hydrocarbures à longues chaînes avec l'augmentation de la température
Sexual behavior in Drosophila is genetically determined. It implies different communication mechanisms (chemical : pheromones; acoustic : song and visual) and simpler behaviors ( like locomotor activity). In addition, it has been demonstrated that terminal enzymes for dopamine synthesis (DDC : dopa decarboxylase and TH : thyrosine hydroxylase) were formed under the cuticle and in the central nervous system. Initially, our research focused on potential differences in cuticular hydrocarbons, linked to the presence or not of dopamine. For that, genetical and pharmalogical approaches were used: TH enzyme inhibitor on wild type strains, mutant strains (Ddc ts) complemented with their lacking compounds and mutant strains for their structural genes: Ddc and pale. We observed a correlation between diminution of dopamine and of female-specific hydrocarbons. Then, we demonstrated that this defect, comparable to the decapitation effect, is compensated for the females by a single topical application of dopamine. We also analysed different behavioral aspects. We demonstrated that male flies having reduced levels of dopamine and serotonine have a lower success rate for copulation, a lower locomotor activity and a modified song. Dopamine seems to play a significant role in sexual behavior regulation at different levels : pheromons production, acoustic production, and locomotor activity. Finally, a study of pheromonal variations as a function of temperature bas been performed. It shows that higher temperatures favor the production of longer chains of hydrocarbons either in males and females in two wild type strains of D. Melanogaster

The present work focuses on the mechanisms of selection for the control of action in Drosophila melanogaster (D. melanogaster), also known as fruit fly. D. melanogaster has a rich repertoire of innate and learned behaviours and a quite simple brain, composed by roughly 100,000 neurons, which can be studied by means of sophisticated techniques. Therefore, it offers the possibility to study complex behaviour in a brain structure simpler than that of higher organisms. As a consequence the neurobiological underpinning of its behaviour can be understood in an easier manner. The comparison of its behaviour with similar behaviours shown by different and evolutively distant animals can provide important insights about their relationship with different or conserved underlying neural circuits. This thesis was conceptualized to sketch out whether selection for action processes underlying the behaviour of mammals might be shared with lower organisms such as D. melanogaster. Selection for action entails a close interaction between visual and motor systems allowing to select a specific stimulus in the environment to which act upon. This process allows to filter out irrelevant information for action. The first experiment was aimed at investigating whether flies have an action-based attention. Are flies able to inhibit via attentional mechanisms the response to an upcoming stimulus in order to successfully end an ongoing action? In particular, I observed whether flies are prone to interference effects caused by the upcoming appearance of a competitive stimulus (i.e., a distractor). I expected this inhibitory mechanism to be played out on spatial trajectories. In this study, flies were engaged in a walking task aimed at reaching a visual target (i.e., a bright stripe) while an abrupt identical distractor was laterally presented. The second experiment pointed at extending the finding of the first experiment. In particular, angular distances between target and distractor were considered. The aim of this study was to test the hypothesis that the shorter the distance between target and distractor the greater is the level of inhibition. Then, in the third experiment I targeted the hypothetical neural circuit underlying the behavioural effects observed in the previous experiments. Based on the increasing evidence for an intriguing homology between a specific neuropil of flies (Central Complex; CX) and the mammals’ neural structure involved in action selection, the idea was to test flies with a lesioned CX during the behavioural task used in the previous experiments. To do this, I used a technique based on the GAL4-UAS binary system in order to downregulate specific dopamine receptors in a very selected neural circuit, the so-called E-PG neurons. Moreover, I adopted an optogenetic technique for in vivo neural manipulation. I employed flies bearing light-sensitive ion channels in the same selected neural circuit of CX to briefly activate such neurons during the task. This neural circuit forms a donut-shape structure which it has been proposed to be an integrative circuit between visual and motor systems and to perform an attention-like function. Finally, in the fourth experiment I characterized a series of neural circuits of CX from a neurochemical perspective. The hypothesis was that the dopaminergic system, involved in the action selection process of mammals, could also modulate the neurophysiological response within the CX of flies. Specifically, I recorded in vivo the neural response to dopamine application in CX of flies by using a bioluminescence technique based on a genetically encoded calcium indicator. All in all, this work represents an attempt to tackle the mechanisms of selection for the control of action in flies. The interference paradigm I developed establishes a powerful platform to further explore the problem of selection for action in flies which might be useful for clarifying similar processes in higher organisms.
Il presente lavoro si focalizza sui meccanismi di selezione per il controllo dell’azione utilizzati da Drosophila melanogaster (D. melanogaster), anche nota come moscerino della frutta. D. melanogaster ha un ricco repertorio di comportamenti e un cervello semplice, composto di circa 100,000 neuroni, che può essere studiato con tecniche raffinate. Perciò, offre la possibilità di studiare un comportamento in una struttura cerebrale semplice rispetto a quella di organismi più complessi. Le basi neurobiologiche del suo comportamento possono essere così più facilmente comprese. Il confronto con comportamenti simili mostrarti da animali evolutivamente più lontani può fornire importanti intuizioni sui circuiti neurali sottesi. Questa tesi è stata concettualizzata per verificare se i processi di selezione per l’azione dei mammiferi potessero essere condivisi con organismi più bassi come D. melanogaster. La selezione per l’azione implica una stretta interazione tra sistema visivo e motorio che consente di scegliere uno stimolo nell’ambiente per agire su di esso. Questo processo permette di filtrare le informazioni irrilevanti per l’azione. Il primo esperimento era finalizzato a indagare se i moscerini mostrano un’attenzione basata sull’azione. Sono capaci di inibire la risposta a uno stimolo grazie a meccanismi attentivi per terminare un’azione? In particolare ho indagato se i moscerini sono inclini all’effetto d’interferenza causato dalla comparsa di uno stimolo competitivo (cioè, un distrattore). Mi aspettavo che questo meccanismo inibitorio fosse evidente nelle traiettorie spaziali. In questo studio i moscerini erano impegnati a raggiungere un target visivo (cioè, una striscia luminosa) mentre un distrattore compariva lateralmente. Il secondo esperimento mirava a estendere le conclusioni del primo esperimento. In particolare sono state considerate le distanze tra target e distrattore. L’obiettivo di questo studio era di verificare l’ipotesi che più corta è la distanza tra target e distrattore, più elevato è il livello d’inibizione. Nel terzo esperimento ho individuato l’ipotetico circuito neurale responsabile degli effetti comportamentali osservati negli esperimenti precedenti. Basandomi su crescenti evidenze in favore di un’affascinante omologia tra un neuropilo dei moscerini (il Complesso Centrale, CC) e la struttura neurale dei mammiferi implicata nella selezione dell’azione, ho testato il comportamento in moscerini con CC danneggiato. A questo scopo ho usato una tecnica basata sul sistema binario GAL4-UAS per ridurre l’espressione di specifici recettori dopaminergici in un circuito neurale molto selettivo, i cosiddetti neuroni E-PG. Inoltre, ho adottato una tecnica optogenetica per la manipolazione neurale in vivo. Ho utilizzato moscerini che esprimevano canali ionici foto-attivabili nello stesso circuito del CC per eccitare tali neuroni durante il compito. Questo circuito neurale forma una struttura a ciambella che è stata considerata un centro d’integrazione tra il sistema visivo e motorio deputato all’attenzione. Infine, nel quarto esperimento, ho caratterizzato da un punto di vista neurochimico una serie di circuiti neurali del CC. L’ipotesi era che il sistema dopaminergico implicato nel processo di selezione dell’azione nei mammiferi potesse modulare anche nei moscerini la risposta neurofisiologica all’interno del CC. Nello specifico, ho registrato in vivo la risposta neurale ad applicazioni di dopamina nel CC dei moscerini utilizzando una tecnica di bioluminescenza basata su indicatori del calcio geneticamente codificati. Nel complesso questo lavoro rappresenta un tentativo di affrontare i meccanismi di selezione per il controllo dell’azione nei moscerini. Il paradigma d’interferenza che ho sviluppato costituisce una potente piattaforma per esplorare il problema della selezione per l’azione che potrebbe essere utile al fine di chiarire processi simili in organismi più complessi.

Les Drosophiles à jeûn peuvent être conditionnées dans le but d’associer une odeur avec du sucre. Un cycle de conditionnement appétitif induit la formation de mémoire à court terme (MCT) et de mémoire à long terme (MLT). Ainsi, nous avons montré que les MCT et MLT sont formées indépendamment l’une de l’autre et impliquent des structures neuronales distinctes au sein des Corps Pédonculés (CPs), le centre de la mémoire olfactive. Nous avons proposé un nouveau modèle de la dynamique des phases de mémoire appétitive où la formation des MCT et MLT s’effectue de manière parallèle. Suite à cette étude, nous avons identifié deux paires de neurones extrinsèques aux CPs impliqués dans la restitution de l’information appétitive à long terme. Enfin, nous nous sommes intéressés aux mécanismes moléculaires et aux réseaux neuronaux impliqués dans la consolidation de la mémoire aversive. Chez la Drosophile, l’appariement d’une odeur à des chocs électriques deux formes de mémoires consolidées, la mémoire résistante à l’anesthésie (MRA) et la MLT (dépendante de la synthèse protéique de novo). Nous avons montré que 3 paires de neurones dopaminergiques aux CPs jouent un rôle «d’interrupteur» contrôlant une bascule entre la MRA et la MLT. Ainsi, bloquer ces trois paires de neurones dopaminergiques durant la période de consolidation induit une augmentation de la MRA et une inhibition de la MLT, alors qu’activer ces neurones après conditionnement entraîne une inhibition de la MRA, et favorise la mise en place de la MLT. En conclusion, nous avons caractérisé fonctionnellement des ensembles neuronaux discrets jouant un rôle dans différentes étapes de l'apprentissage et de la mémorisation olfactifs
When we present an odor associated with sugar to starved flies, they will be attracted by this odor. One cycle of conditioning induces both Short-Term Memory (STM) and Long-Term Memory (LTM). It is accepted that STM and LTM formation is a sequential process but the link between these two memories remains unknown. We adressed this question and clearly demonstrated that STM and LTM can be formed independently and that they involved different neural structures within the Mushroom Bodies (MB), a memory center. We proposed a new model of dynamic of appetitive memory phases where STM and LTM are formed in a parallel way. Then, using the genetically expressed thermosensible toxine allowing a transiently inactivation of neurotransmission, we identified one type of MB efferent neurons involved in appetitive LTM retrieval. Additionally, we were interested to the molecular mechanisms and the neuronal circuits involved in aversive consolidated memories. Pairing an odor with electric shocs induces aversive memory. In drosophila, there are two forms of consolidated memories, the Anesthesia-Resistant Memory (ARM) and LTM (dependent on de novo protein synthesis). We show that three pairs of oscillatory dopaminergic neurons play a essential role of gating between ARM and LTM formation. So, blocking the neurotransmission of these neurons during the consolidation phase leads to a increase of ARM and inhibition of LTM whereas, artificial activation of these neurons after conditioning leads to an inhibition of ARM and favors the implementation LTM. In conclusion, we characterized functionally a restricted population of neurons playing a role in various stage of learning and memory process

The transport of organic cations (OCs) is important for lowering an organism’s susceptibility to the toxic effects of endogenous and exogenous OCs. Endogenous OCs include choline and N¹-methylnicotinamide (NMN) and catecholamines, while exogenous OCs may include xenobiotics, such as drugs, pesticides, and environmental toxins. These potentially toxic compounds must be eliminated from the organism to ensure survival. The midgut and Malpighian tubules of insects have been shown to be involved with the active transport of OCs. Two putative organic cation-like transporters, named orct and orct2, have previously been identified and cloned from adult Drosophila melanogaster. Previous quantitative real-time polymerase chain reaction studies showed mRNA transcripts of orct and orct2 were differentially expressed in the midgut and Malpighian tubules, and expression patterns increased following exposure to prototypical type I OC, tetraethylammonium (TEA). These findings suggest that the recently cloned Drosophila orct and orct2 will function as a transport protein for the OC substrate TEA. In this study, a dual-function expression vector, pXOOM, was used to design a molecular construct for the heterologous expression and functional characterization of Drosophila ORCT in Xenopus laevis oocytes. The kinetic profile of the expressed ORCT for TEA was determined using [¹⁴C]-labeled TEA assays. Analysis of the TEA functional assays revealed that insect ORCT is capable of a saturable, carrier-mediated transport of TEA. Maximal transport capacity (Jmax) and transport affinity for mediated TEA uptake (Kt) were 5 µmol L-¹ per oocyte and 0.33 mmol L-¹, respectively. ORCT-mediated TEA uptake was inhibited in the presence of type I and type II OCs. Quinine and verapamil inhibited TEA influx by 33 and 43%, respectively, whereas cimetidine and vinblastine did not reduce TEA uptake. These experiments coincide with previous physiological research on OC transport across insect renal tissues. The knowledge gained from this thesis may provide the basis for the development of an effective and environmentally benign insecticide, as well as allow a greater understanding of the deleterious effects of environmental pollutants on insect populations.
Irving K. Barber School of Arts and Sciences (Okanagan)
Biology, Department of (Okanagan)
Graduate