Univ. Prof. Dr. Harald Sitte

Project I - Pharmacological characterization of fluorescent and / or optical probes for neurotransmitter transporters

Background / Objective: Neurotransmitter transporters serve as clinically relevant targets for the treatment of depression and attention deficit hyperactivity disorder. Hence, the development of compounds targeting monoamine and also the organic cation transporters which mediate the retrieval of neurotransmitters from the synaptic cleft. The student will characterize (i) novel fluorescent dyes established for the imaging of various neurotransmitter transporters such as organic cation transporters 3 and dopamine transporters (collaboration with the Maulide group) and (ii) optically active probes that change their conformation upon illumination with specific wavelengths which will be developed as tool compounds for testing conformational equilibria (collaboration with the Mihovilovic group). The compounds will be examined pharmacologically by using various biochemical approaches. This will fuel the in silico approaches performed in the Ecker group, with direct repercussion onto the compound development. In parallel, we will use fluorescence microscopy to unveil whether the compounds properly bind to the surface-expressed transporters. Subsequently, we will assess the conformational equilibrium of transporters by fluorescence resonance energy transfer (FRET) microscopy to understand the impact of the binding and monitor conformational changes, also by electrophysiology. In addition, the student will test whether the compounds influence the well-established oligomeric properties of these transporters: A number of psychoactive compounds have been shown to disrupt oligomer formation. However, it is thus far unclear how these compounds elicit these effects. One possibility might be via a disruption of the intimate interaction with plasma membrane lipids, the phosphoinositides. Since we have recently shown that the quaternary transporter structure depends on the content of those lipids in the plasma membrane of the living cell, we will examine phosphoinositide-analogs developed by the Mihovilovic group. The planned measurements will be accompanied by mass spectrometry to reach a quantitative level.

Methods: Methods employed by the student will encompass radiotracer flux measurements (influx, efflux), fluorescence microscopy including FRET microscopy, electrophysiology as well as the probe generation for mass spectrometry. Manipulations of the plasma membrane content will be achieved by inducing the activity of phospholipase C by well-established activators.

Networking: As outlined above, the project is a collaborative effort of the Sitte laboratory with the Ecker, Maulide and Mihovilovic laboratories. All biological and pharmacological examinations will be performed in the Sitte group; the expertise in the area of docking and homology modelling by the Ecker group will be necessary to design novel mutants and interpret the results in a structural context, which will ultimately fuel the efforts in the chemistry laboratories of Maulide and Mihovilovic.

Expected results: Ultimately, the student will establish fully characterized novel fluorescent ligands for neurotransmitter transporters and learn about their effects on the conformational equilibrium and quaternary arrangement.

Project II - The impact of oligomerization on organic cation transporter 3

Background / Objective: It has been shown that organic cation transporter (OCT) isoforms 1 & 2 form quaternary structures in the living cell. Quaternary structures, however, have been intricately linked to the ability of amphetamines to induce efflux via monoamine transporters. Since the knowledge regarding the oligomeric state of OCT3 is sparse. However, most recently, we have shown that OCT3 is also impacted by this class of psychoactive compounds. Hence, in this subproject we will examine the propensity of OCT3 to form oligomeric structures in living cells by using human embryonic kidney cells as well as rat pheochromocytoma 12 (PC12) cells. PC12 cells endogenously express OCT3 and are therefore ideally suited to test the hypothesis by using the compound developed under Aim 1 (collaboration with the Maulide group). HEK293 cells do not express OCT3 and therefore, we will use genetically encoded fluorescent markers to visualize the transporters at the plasma membrane of the cells. Subsequently, we will apply FRET microscopy to establish whether the transporters are in close proximity. Finally, we will elucidate the pharmacological impact that quaternary organization of OCT3 transporters plays in both paradigmatic cell lines: To achieve this, we will use classical biochemical and pharmacological experiments to characterize functionality of the transporters. In order to establish whether OCT3 forms oligomeric structures via distinct interaction interfaces, the student will examine possible contact domains, apply mutagenesis and test whether those mutated transporters display disrupted oligomeric properties. These resulting transporters will be assessed on their functional possibilities with special emphasis to the action of amphetamines.

Methods: Methods employed by the student will encompass radiotracer flux measurements (influx, efflux) in addition to fluorescent tracers, fluorescence microscopy including FRET microscopy, electrophysiology as well as molecular biology to create fluorescently tagged transporters.

Networking: As outlined above, the project is a collaborative effort of the Sitte laboratory with the Maulide laboratory. All biological and pharmacological examinations will be performed in the Sitte group; fluorescently labeled corticosterone, a specific inhibitor of OCT3 will be synthesized in the Maulide laboratory.

Expected results: Ultimately, the student will fully characterize the novel fluorescent ligands for OCT3 and learn about their functional effects. Furthermore, the student will assess the oligomeric properties of OCT3 and learn about possible influencing factors. Subsequently, the student will be able to assess the potential impact of oligomerization on inwardly directed transport or on efflux.