Fluorescence spectroscopy of the serotonin transporter

Specific transporter proteins localized in the presynatic nerve terminals are responsible for the rapid removal of released neurotransmitters, such as serotonin, dopamine and norepinephrine, from the synaptic cleft. In addition to play a key role in regulating the availability of the transmitters in the synaptic cleft, these proteins are targets for the action of many drugs. This includes both antidepressants and widely abused psychostimulants, such as cocaine, amphetamine, and `ecstacy`. Despite the physiological and pharmacological importance of the transporter proteins surprisingly little is known about how they work at the molecular level. Using the human serotonin transporter (hSERT) as a model system, it is the overall goal of the present project to understand the structural basis for the ability of Na+/Cl--dependent neurotransmitter transporters to translocate their substrate across the plasma membrane. In particular, we will focus on delineating the structural basis and molecular significance of transporter oligomerization. Notably, the applicant has most recently been able to provide the first direct evidence that hSERT exists in an oligomeric state in living cells. The research strategy employs the integrated use of molecular biology and fluorescence spectroscopic methods. First, we will seek to investigate the functional significance of oligomerization by quantatively and qualitatively assess oligomerization directly in living cells under different functional conditions. Next, it is the goal to define the structural organization of the oligomeric complex by the application of fluorescence spectrscopy relying on resonance energy transfer (RET). Finally, it is the aim to identify both intra- and intersubunit structural changes accompanying translocation of serotonin across the plasma membrane. It is our expectation that the studies not only should provide new paradigms regarding the molecular function of Na+/Cl--dependent neurotransmitter transporters but also unravel new putative strategies for treatment of psychiatric diseases and drug abuse.