- Project 1: Molecular insights into the role of lipids and regulatory proteins on sodium
- Project 2: NMR based structural and dynamic investigation of the transport cycle of NSS transporters
- Project 3: Purification, functional characterization and reconstitution of DAT and LeuT
- Project 4: Development of a comprehensive model of GABA transporter function
- Project 5: Electrophysiological characterization of neurotransmitter transporters and formulation of an overall kinetic model
- Project 6: Electrophysiological approaches to study ion coupling and transport by NSS
- Project 7: Structure and kinetic mechanism of neurotransmitter transporters
- Project 8: Develop novel approaches to measure transporter-ligand interactions
- Project 9: Integrative modelling of ligand binding, transporter stability and the transport cycle
- Project 10: Time resolved structural studies of membrane
- Project 11: Single-molecule studies of secondary-active transporters and their environmental dependence
- Project 12: Resolving coarse-grained dynamic distance constraints of the transport cycle of NSS transporters using EPR spectroscopy
- Project 13: Characterization of the molecular mode of action of novel psychoactive substances
- Project 14: Development of a microfluidic cell perfusion station for controlled compound delivery and real time fluorescence monitoring
- Project 15: Assessing the molecular determinants of novel disease causing mutations in DAT
Project 3: Purification, functional characterization and reconstitution of DAT and LeuT
The neurotransmitter transporters (NSSs) are membrane proteins located to the presynaptic terminal on neurons. They control neuronal communication by rapid reuptake of neurotransmitters. The rewarding effects of cocaine is due to its inhibition of the NNS for dopamine (DAT). There is no medical treatment for cocaine addiction. The atypical DAT inhibitors (ADIs) do also inhibit DAT, but they are not rewarding. Rather, they antagonize the effect of cocaine, making ADIs possible cocaine antidotes. The mechanism behind this is largely unknown, and the further exploration of this conundrum has been hampered by experimental limitations in the purification of functional DAT. We are the first to report the purification of functional DAT wild type.
The purpose of this project is to elucidate the fundamental differences in binding dynamics between cocaine and ADIs with advanced pharmacological technologies and state-of-the-art biophysical methods. This could include structural determination. We will find the molecular determinants that constitute the distinct pharmacological signatures of ADIs and cocaine. Our profound expertise in investigating protein conformational dynamics allows us to elucidate the specific DAT conformations induced by the two drug classes. Specifically, we will measure distances between specific DAT domains and how they are influenced by ligands. We wish to map the global structural dynamics induced by ligand binding using e.g. FRET-based methods and unnatural amino acids. Together, they could reveal a conformational ‘fingerprint’ for each drug class enabling a prediction for categorizing novel compounds.
We expect that these findings will provide novel information on complex conformational dynamics on a hitherto unpreceded level of detail and provide fundamental insight in why some DAT inhibitors are stimulants while others are not. This will contribute significantly to the development of a drug against cocaine abuse.
