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.