Characterization of a mutant dopamine transporter in HEK-293 cells
Abstract
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.