The paramount importance of human membrane proteins for life contrasts with the lack of understanding of their molecular mechanisms of function and pharmacology. This project focuses on the molecular mechanisms of one of the most important and medically relevant families of neurotransmitter membrane transporters in the brain, the Human Excitatory Amino Acid Transporter (hEAAT) family. Glutamate is the main excitatory neurotransmitter in the brain and its extracellular concentration is mainly regulated by the hEAATs, which pump the transmitter into the cytoplasm of cells. This role is essential to neurological processes like memory, cognition, and learning. Importantly, glutamate can also be a potent neurotoxin and the deregulation of its extracellular levels is associated to numerous neurodegenerative and mental disorders. We aim to characterize the molecular function and pharmacology of the hEAATs using a broad multidisciplinary biophysical approach to obtain and integrate structural, energetic and dynamic information on these human membrane proteins. We will develop new methods to obtain for the first time the hEAATs in pure and stable form for rigorous biophysical analysis and generate a library of new selective compounds with pharmaceutical potential that target the transporters. This knowledge will be essential to gain a complete understanding of hEAATs function and to design new therapeutic and pharmacological strategies to alleviate the impact of common neuropathologies. Finally, the methods that we will develop for the hEAATs could be applied to other families of human membrane proteins and will help to bring our knowledge on membrane proteins to the human level.