Mechanism of Intracellular Gating in the Glutamate Transporter

Abstract

Glutamate transporters (GlTs) are membrane proteins that regulate and remove synaptically released neurotransmitter glutamate, and maintain normal excitatory synaptic transmission. The recently solved structure of inward-facing GltPh, a GlT homologue revealed an occluded state with the substrate and two Na+ ions (Na1 and Na2) bound. The inward-facing and outward-facing structures of GltPh have put forward a molecular mechanism by which the transporter mediates Na+-coupled substrate uptake. However, the molecular nature of the intracellular gate and the mechanism of gating are still unknown. Furthermore, the mechanism of release of the substrate and co-transported Na+ ions from their intracellular binding sites remains elusive. We have investigated the transporter's dynamics and the coupling between substrate and Na+ ions using an extensive set of molecular dynamics simulations of membrane-embedded model of inward-facing GltPh in various bound states. The results suggest that the helical hairpin HP1 plays the key role of the intracellular gate for the substrate-binding site, and that the opening and closure of the gate is controlled by the Na+ ion in the Na1 site. The Na+ ion in the Na2 site was found to be the first to be released from the inward-facing occluded state and can diffuse into the cytoplasmic solution through the attraction of highly conserved residue Ser65 in TM2. Moreover, upon unbinding of the Na+ ion in the Na1 site, the substrate was observed to completely unbind from the binding site and diffuse into the cytoplasmic solution in our equilibrium simulations along the opening of the intracellular gate HP1. Based on the simulations, we propose that the two structurally resolved Na+ ions release into the cytoplasm froe the inward-facing Gltph before the substrate.