Paran Davari


Psychostimulants such as amphetamine and methamphetamine target dopamine transporter (DAT). The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. It has been shown that amphetamine and methamphetamine differentially alter the activity of DAT, although, it is unknown whether amphetamine versus methamphetamine interaction with DAT, differentially stabilizes the transporter in a specific membrane microdomains. In my research, I use a fluorescent microscopic approach to test the hypothesis that DAT association with specific microdomains in the plasma membrane is differentially altered by amphetamine vs. methamphetamine exposure. In my experiments, I use membrane raft marker cholera toxin subunit-B (CTxB) CTXB labeling. CTXB pentavalently binds to GM1 sphingolipids, resulting in co-clustering of raft-associated proteins. I predict to measure even distribution of YFP-DAT in the plasma membrane in untreated cells. Staining of the cells with Alexa-647-conjugated CTxB (Alexa647-CTXB), or CFP-TFR will result a punctuate localization at the surface membrane. I also want to measure a partial co-localization of YFP-DAT/Alexa647-CTxB and also a partial co-localization CFP-TfR/DAT. By performing these experiments, we can determine whether amphetamine versus methamphetamine influence membrane microdomain distribution of DAT. In long-term, I intend to examine the functional consequence of membrane microdomain distribution of DAT.