Glutamate is the primary excitatory neurotransmitter in the human brain, responsible for cognition, memory formation, learning, and pain signaling, among other functions. Two families of integral membrane glutamate transporters are essential players in glutamatergic neurotransmission. The VGLUT family loads glutamate into the synaptic vesicles, and the EAAT family removes the neurotransmitter from the synaptic cleft following neurotransmission. The dysregulation of these transporters under pathologic conditions and neurologic disorders disrupts glutamate homeostasis leading to aberrant neurotransmission, glutamate excitotoxicity, and neuronal death. Ions play crucial roles in the function of these transporters. Electrochemical gradients of ions power the concentrative glutamate uptake and regulate transporters. In addition, transporters can conduct ions in a manner uncoupled from the neurotransmitter uptake, modulating electrochemical trans-membrane gradients. This grant proposal aims to understand the mechanism and evolution of ion-coupling mechanisms, mechanisms of the uncoupled ion permeation in health and disease, and ion-mediated regulation. We will combine bioinformatics, single-particle cryo-electron microscopy, single-molecule fluorescence microscopy, and other biophysical and biochemical approaches to pinpoint the residues, the conformational states, and the dynamic properties of transporters underlying their interactions with ions.