Characterization of a mutant dopamine transporter in HEK-293 cells
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Recently, research has demonstrated potentiation of the dopamine transporter’s function by ethanol. This, coupled with data showing that the modulation of transporter uptake is determined by changes in trafficking and not function, implied that ethanol increases the number of transporters on the cell surface. To identify which amino acid sites may be targets for ethanol, hybrid transporters were constructed that share different ratios of amino acid sequences with the dopamine transporter and the norepinephrine transporter, a similar protein that lacks ethanol-mediated potentiation. Dopamine transporter mutants were created at the four most promising amino acid residues using site directed mutagenesis. Two mutants expressed in Xenopus oocytes showed some sensitivity to ethanol and two did not. The quadruple transporter mutant, which contains all four amino acid mutations, demonstrated the most interesting phenotype of inability to take up dopamine. The present study confirms this lack of function in human embryonic kidney (HEK) cells, a mammalian expression system, and addresses whether or not the IGLF dopamine transporter is on the surface, yet not functional, or is simply not trafficked to the cell surface. To accomplish this, HEK cells stably expressing the wild-type or quadruple mutant transporter were created. Radioactive uptake assays were used to determine the extent to which each cell type was able to take up dopamine. Cell surface biotinylation and Western blots were then used to identify surface transporters Both preliminary results and the current study showed lack of uptake in HEK cells expressing the mutant, while the latter determined that the mutant protein was present on the cell surface, albeit to a lesser extent than wild-type protein. By demonstrating that the transporter is indeed being trafficked to the surface, the implication that the mutant is simply not functional as a dopamine transporter becomes the more likely possibility.