This dissertation consists of three parts. The first part explores the possibility of screening a functional but cocaine insensitive norepinephrine transporter and generation of a cocaine insensitive knock-in mouse line, the second part attempts to identify residues in mouse norepinephrine transporter (NET) transmembrane domain 3 (TMD3) that would differentiate norepinephrine and dopamine uptake, while the third part discusses that two extracellular cysteines in dopamine transporter form a disulfide bond, which is vital for the transporter’s function.
Cocaine blocks dopamine transporter (DAT), NET and serotonin transporters (SERT) in the brain and increases extracellular neurotransmitter concentration in various brain regions. It is not known how each of these contributes to complex cocaine addictive effects. Genetically modified mice with a single one of these transporter removed still prefer cocaine, suggesting that none of them is absolutely required for cocaine rewarding effect. We have generated one unique knock-in mouse line, carrying a cocaine insensitive DAT. This mouse line does not show cocaine preference when given cocaine, which showed that DAT is necessary for cocaine rewarding effects. However, how NET is involved in cocaine addictive effect is still unknown. Based on the previous study, I performed several round of random mutagenesis around residues F105 and F150 in transmembrane regions 2 and 3 region. One triple mutation (F101C-A105G-N153T, mNETCGT) that retains wild type uptake activity for substrates but displays decreased cocaine affinity by 37 fold was found. Interestingly, this mutation also decreases desipramine affinity by 24 fold. These results reveal a number of residues in transmembrane regions 2 and 3 that are important for inhibition by different drugs.
To study the exact role of NET involved in cocaine addiction, a knock-in mouse line carrying the above functional and cocaine insensitive NET was generated by replacing wild type NET with mNETCGT. Feeder cell free ES cells (E14Tg2A) were introduced to generate a unique mouse line. This mouse line will be used to study the role of NET in mediating the addictive action of cocaine and the therapeutic effect of desipramine.
Recent research revealed that NET also plays an important role in the regulation of DA homeostasis. Hence, delineating residues of NET differentially involved in DA and NE uptake would provide potential intervention for treatment of drug abuse, depression or other related psychiatric disorders. Our study identified for the first time residues in the TM3 region of mNET that are more critical for NE uptake than DA.
Dopaminergic neurotransmission is terminated by removal of extracellular dopamine via DAT, which belongs to a Na+/Cl- dependant neurotransmitter superfamily. Information about the structure and function relationship of transporters allows us to better understand the molecular mechanism of these transporters and therapeutic drug design. Cysteine residues in transporters form inter- or intra- molecular disulfide bonds which may be critical for proper protein folding, trafficking, surface expression, stability and uptake function. Replacing two extracellular cysteines with alanine in Drosophila melanogaster DAT (dmDAT) abolished transporter uptake activity and surface expression. It has been proposed that these two cysteines form a disulfide bond. However, there was no evidence for the existence of such a disulfide bond. Thus, Dr. Gu generated one functional dmDAT mutant with all cysteines replaced by other residues except two extracellular loop cysteines (EL2), and this mutant was analyzed for this EL2 disulfide bond function.