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UBC Theses and Dissertations

Functional characterization of heterologously expressed Drosophila melanogaster organic cation transporter ORCT in Xenopus laevis oocytes Cruz, Melissa Joy


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.

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