Analysis of whole cell lysates by immunoblotting with anti-C-terminal DAT antibody (Fig. 2A bottom portion) showed complete absence of mature glycosylated DAT (85 kDa) in mutants Gly386Arg and Tyr470Ser.
Torres et al. [10] found that the C-terminal of DAT was not essential for oligomerization, and that a small fragment comprising the first two TMDs inhibited the wild-type transporter function but not when the leucine repeat motif present in the 2nd TMD was mutated.
The total lysates and biotinylated proteins were resolved on 8% Tris-glycine mini gels and probed with polyclonal anti-DAT antibody against the C-terminal of DAT (Millipore), before horseradish peroxidase-conjugated goat anti-rabbit antibody.
A slow delivery to the plasma membrane was also observed in the C-terminal mutant DATs [27].
Dopaminergic lesions result in the loss of DAT terminals through degeneration, but there are additional losses in DAT function that result from a simultaneous reduction of DAT export, leading to its accumulation in the ER Golgi compartment.
One common area for their interaction is the nerve terminal, including dopamine transporter (DAT) systems.
This suggested that Mn may affect the presynaptic dopaminergic neuronal terminal through modulation of DAT function and levels.
In addition, the DAT N-terminal domain has been structurally modeled to identify the locations of positively charged patches that interact with negatively charged signaling lipids [phosphatidylinositol 4,5-bisphosphate (PIP2)].
Blots were immunostained overnight at 4 °C with the following primary antibodies: anti-Tyrosine Hydroxylase (Santa Cruz Biotechnology #SC14007, 1 2000 dilution), N-terminal rat anti-DAT (Chemicon #MAB369, 1 500 dilution) and mouse anti-Actin (Sigma-Aldrich #A2853, 1 20000 dilution) and immunostained overnight at 4 °C with primary antibodies.
These results demonstrate that the presence of the N-terminal tag leads to impaired DAT protein expression in vivo due in part to improper trafficking of the tagged transporter, and highlight the importance of the N-terminus in the transport of DAT to striatal terminals.
Here, we report the development of a new BAC transgenic mouse line that expresses DAT with an N-terminal HA-epitope (HAD-Tg).
