Excitatory amino acid transporters (EAATs) are crucial for clearing synaptic glutamate, and their dysfunction is implicated in a range of devastating conditions, including neuropsychiatric disorders like schizophrenia and neurodegenerative diseases such as Alzheimer's, Parkinson's diseases. These transporters utilize electrochemical gradients of Na + , H + , and K + to drive large-scale conformational changes between outward-facing (OF) and inward-facing (IF) states. While the general mechanism is known, especially the precise molecular role of K + in facilitating the key IF-to-OF transition remains poorly understood. Here, we elucidate this mechanism using a powerful multi-stage computational strategy based on all-atom molecular dynamics simulations with an aggregate simulation time exceeding 50 microseconds. Our approach integrates on-the-fly probability enhanced sampling (OPES) to identify the cryptic K + binding site, steered MD (SMD) to induce a transition between IF and OF stets, optimized nonequilibrium MD simulations to generate realistic transition pathways, and extensive bias-exchange umbrella sampling (BEUS) to map the complete free-energy landscapes. These simulations were guided by a novel set of system-specific reaction coordinates that capture the essential translational and rotational motions of the transport domain. By directly comparing the free-energy landscapes of the transporter in its K + -bound and apo states, we provide definitive evidence for the ion's role. Crucially, our results show a significant free energy barrier and difference for the IF-to-OF transition in the absence of K, which is lowered upon K + binding. This demonstrates that K + is not merely a passenger but an essential co-factor that mechanically enables the return of the transporter to the outward-facing state, ensuring the efficiency of the transport cycle.
Fakharzadeh et al. (Sun,) studied this question.