Synaptic uptake of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine is mediated through transporters situated in the presynaptic membrane, thereby terminating the action of these molecules. These closely related transporters, DAT, SERT, and NET, respectively, are important targets for many psychotropic drugs, including cocaine, antidepressants, and amphetamine. We have used a confocal single molecule fluorescence spectroscopy technique, fluorescence correlation spectroscopy (FCS), to asses directly the mobility of YFP-tagged DAT in living cells. In both stably and transiently transfected HEK-293, HeLa and AN27RB3 cells we obtained diffusion times for membrane localized DAT around 20 ms, consistent with a freely diffusible membrane protein. Similar diffusion times were observed for the homologous GABA transporter-1 tagged with YFP in these cell types. In contrast, our FCS measurements in transiently transfected N2A neuroblastoma cells were impaired by photobleachning suggesting immobilization of the transporter in the membrane. This was confirmed by the use of fluorescence recovery after photobleaching (FRAP), which showed clear recovery of YFP-DAT fluorescence in HEK-293, HeLa and AN27RB3 cells but little or no recovery in N2A cells. Interestingly, the restricted mobility of YFP-DAT in N2A cells was partly reversed with the addition of methyl- -cyclodextrin, a cholesterol depleting agent, suggesting that YFP-DAT is sequestered in lipid microdomains. In summary, our data represent the first assessment of microdomain localization of the dopamine transporter and demonstrate FCS as a new and useful tool for studying mobility of membrane proteins in general.
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Society for Neuroscience 33th annual meeting, 2003