The aspartate transporter from Pyrococcus horikoshii, Glt_Ph, is a concentrative solute transporter homologous to human glutamate transporters. Glutamate transporters use steep gradients of Na⁺ to sequester glutamate within presynaptic neurons and glial cells to prevent toxic accumulation of glutamate at the excitatory synapse. X-ray crystallography provides structural details of Glt_Ph, while functional data is obtained by reconstituting the transporter in liposomes.

There currently exists a collection of high-fidelity crystal structures that have captured Glt_Ph in a variety of states representative of the transport cycle. These structures have revealed Glt_Ph to be trimeric, with the binding site for aspartate held between two re-entrant hairpin loops (HP2 and HP1), the extracellular and (putative) intracellular gates, respectively (Boudker et al., 2007; Reyes et al., 2009). The final structure required to complete the conformational ensemble of Glt_Ph is with substrate-bound and HP1 propped open, poised to release substrate.

When reconstituted into liposomes, Glt_Ph distributes as a random population of inward- and outward-facing transporters, which can obscure subtle transporter kinetics. A novel approach using MTS-reagent thiol modification (Tsai et al., 2012) has allowed the development of sided-inhibition of Glt_Ph to investigate oriented kinetics of the transporter. Using this strategy, the inhibitor profile of a variety of N-modified aspartate analogues has been characterised and are shown to have preference for the inward-facing orientation of Glt_Ph. This preference for the ‘intracellular’ binding site allows us to probe the molecular determinants of inhibitor affinity and may provide an avenue to ‘lock’ the transporter in a state amenable to X-ray crystallography.