Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold.
Using single moleucle FRET, we have directly observed these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.
Transport domain unlocking sets the uptake rate of an aspartate transporter. Akyuz N, Georgieva ER, Zhou Z, Stolzenberg S, Cuendet MA, Khelashvili G, Altman RB, Terry DS, Freed JH, Weinstein H, Boudker O, Blanchard SC. NATURE (2015) 518(7537):68-73.
Transport dynamics in a glutamate transporter homologue. Akyuz N, Altman RB, Blanchard SC, Boudker O. NATURE (2013), 502(7469):114-118.