Molecular and Cellular Analysis of
Dopamine and Serotonin Transporters
Dear Dr. Lee, you show very interesting results on the co-transfection of cells with DAT+D2short, which strongly increased the functional DA transport, as assessed by 3H-DA uptake. You conclude that the effect of D2short was independent of agonist stimulation, based on the data with quinpirole. However, in those experiments, cell preincubated with quinpirole (1-10 uM) were then exposed to 3H-DA (1-50 uM), which is an agonist at the D2short receptor. Could it be that you don't see any effect of quinpirole just because the receptor are fully activated by the DA used for uptake experiments? In such case the data with the antagonist (which one was used, and what concentration?) could be more indicative of the possible involvement of receptor activation for increasing DA uptake.
A second point that I would like to discuss with you is the relation between the functional experiment and the confocal microscopy picture (very nice!): in the latter experiment the cells were not pre-incubated with DA or quinpirole, therefore you just see that the co-expression of D2short changes the subcellular distribution of DAT from the cytoplasm to the membrane.
Did you measure total DAT protein (using antibodies and/or ligand binding experiments) and DAT mRNA after co-transfection?
Knowing if the co-transfection increases DAT protein or simple change its subcellular distribution could be of help in understanding if D2short acts only as 'chaperon' molecule or if it modulates the expression of DAT. With many thanks, Tiziana Mennini
Fri Dec 11