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.