- 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 6: Electrophysiological approaches to study ion coupling and transport by NSS
The aim of this project is to comprehend how sodium electrochemical gradient energizes the translocation of the substrates in the various steps of the cycle of GABA transporters and define in these states the role of chloride. The steps of the transport cycle of NSS proteins are associated with movements of charges (co-transported or intrinsic) through the membrane electric field. The sodium ions and substrates interaction in each step will be investigated by examining the currents (pre-steady state, leakage and steady-state currents) recorded in different conditions: altering driving force by varying intra- and extra-cellular concentrations of Na+ and Cl-, changing substrate and inhibitors, temperature, pH and voltages.
The biophysical data will be integrated with the uptake data: acquiring, in the same experiments, in a radiolabelled free approach, the amount of substrate entered the cell. These data allow to define electrogenicity and transport stoichiometry. This study will help to develop a complete model of the transport cycle in the presence of the substrate and of the inhibitors. Wild type, mutants, and chimeric GABA transporters will be analysed.
Mutants and chimeric protein will be prepared according to the prediction of the models of the project 4 and 9 or proposed by structural knowledge from project 1. Determinants considered essential in binding and moving ions and substrates across the membrane will be generated and investigated. Moreover, in this project, some other SLC6 proteins will be studied in parallel because NSSs share the transport mechanics while differ in co-transported ion stoichiometry and in chloride dependence. Data about mutants will be collected in collaboration with project 5 and support the project 13.
The main objectives of Project 6 are:
- determine the stoichiometry of the GABA transporters, developing a radiolabeled free method coupled to electrophysiological recording
- define the steps, rate and kinetic parameters of the transport cycles, investigating ion and substrate binding under vary conditions as described before and compare to experimental data of Project 4 and 5.
- Functional identification of the ion binding sites and of the residues involved in the main steps of the transport cycle, supporting the indications of Project 4.
- identify general valid principles through the comparison between the NSS transporters. Data validation will be carried out by comparison to data obtained in Projects 1, 4, 5, and 11.
