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

摘要

Excitatory amino acid transporters (EAATs) harness [Nasup+/sup], [Ksup+/sup], and [Hsup+/sup] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Nasup+/sup ions, [Nasup+/sup] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Nasup+/sup:substrate coupling in the EAAT homolog GltsubPh/sub. A lipidic cubic phase x-ray crystal structure of wild-type, Nasup+/sup-only bound GltsubPh/sub at 2.5-? resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Nasup+/sup 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 Nasup+/sup-stabilized gate opening and electrostatic coupling of aspartate to Nasup+/sup binding provides a constant Nasup+/sup:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.