In the current study, we aimed to introduce tetraCys residues in DAT extracellular domains that intrinsically encode Pro-Gly dipeptide sequences at DAT amino acid positions 194, 288 and 387. The CCPGCC sequence is the optimal sequence to facilitate biarsenic dye binding, and once bound confers the proper dye conformation for fluorescence emission 
. Converting the four residues flanking the Pro-Gly at amino acid positions 194–195 yielded a DAT that retained DAT surface expression comparable to wildtype. Moreover, EL2-CCPGCC DAT exhibit robust DA uptake, albeit somewhat reduced as compared to wildtype, and retained sensitivity to PKC-mediated downregulation, suggesting that this mutant may be amenable to labeling with biarsenic dyes for live imaging studies of DAT trafficking. A recent study indicated that inserting a tetraCys motif at the DAT amino terminus and at position 511 were also well tolerated 
. However, these are both located on intracellular domains that would require use of a membrane permeant biarsenic dye that would have access to the entire cellular DAT population and would not exclusively label the DAT surface residents. It is also interesting to note that although we reproducibly observe PKC-mediated DAT downregulation in PC12 cells, a recent study by Ericksen et al reports lack of PKC-mediated downregulation in primary ventral midbrain neurons prepared from P2 rat pups 
. This conflicts with previous studies that report PKC-mediated DAT downregulation in adult rat striatal synaptosomes 
, and suggests that either the developmental stage of the dopaminergic neuron or the primary culture environment may influence the ability of DAT to undergo PKC-mediated downregulation.
While our data demonstrate that an extracellular tetracysteine-tag is well tolerated by DAT, they do not demonstrate whether this DAT construct can be labeled with biarsenic dyes. We made several attempts to label EL2-CCPGCC DAT using Lumio brand (Invitrogen) FlAsH reagent, using both standard approaches as well as modified protocols that use extended washes with BAL reagent to reduce non-specific FlAsH binding. All of our labeling attempts resulted in a high degree of non-specific labeling of non-transfected cells, as well as cells expressing either wildtype or EL2-CCPGCC DAT (not shown). Problems with non-specific FlAsH binding have been described 
and particular issues have been raised regarding the utility and specificity of the commercially available Lumio form of FlAsH 
. Thus, while our EL2-CCPGCC DAT is a viable DAT form, advances in FlAsH labeling may be required in order to determine the utility of this mutant in future trafficking studies.
DAT residues 192–197 are located between the second and third glycosylation sites of the large second extracellular loop of the protein. This region appears to tolerate amino acid substitution well, as previous studies reported that an extracellular HA epitope was successfully engineered across residues 193–203 
. The HA epitope at this locus is accessible for extracellular antibody labeling, suggesting that the much smaller biarsenic dye would also have access to this DAT region. Cysteine conversion of EL3 and EL4 residues flanking the Pro-Gly at positions 288–289 and 387–388, respectively, yielded CCPGCC DAT mutants that were not highly functional. Interestingly, a previous mutagenesis study in which a cysteine-depleted DAT was generated indicated that mutating an existing cysteine in EL3 at position 306, just downstream of the EL3-CCPGCC DAT, resulted in the expression of mature DAT protein 
that is fully functional 
. Cys306 is positioned close to the sixth transmembrane segment, suggesting that this region may be more tolerable to structural perturbation.
In summary, we have characterized a functional DAT mutant that encodes a tetraCys motif on the extracellular DAT domain EL2. The ability to introduce cysteines at this locus may have future utility both for NTSEA reagent labeling, as well as for biarsenic dye labeling for live imaging studies. Future studies will directly examine the capacity of this mutant to bind to biarsenic dyes.