Na⁺-dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters

Excitatory amino acid transporters (EAATs) harness [Na⁺], [K⁺], and [H⁺] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na⁺ ions, [Na⁺] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na⁺:substrate coupling in the EAAT homolog Glt_Ph A lipidic cubic phase x-ray crystal structure of wild-type, Na⁺-only bound Glt_Ph at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na⁺ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na⁺-stabilized gate opening and electrostatic coupling of aspartate to Na⁺ binding provides a constant Na⁺:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.