title: Opposite movement of the external gate of a glutamate transporter homolog upon binding cotransported sodium compared with substrate.
abstract: Recently, a new model for glutamate uptake by glutamate transporters was proposed based on crystal structures of the bacterial glutamate transporter homolog Glt(Ph). It was proposed that hairpin two (HP2) functions as the extracellular gate and that Na(+) and glutamate binding closes HP2, thereby allowing for the translocation of the glutamate binding pocket across the membrane. However, the conformation of HP2 in the apo state and the Na(+) bound state is unknown. We here use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter Glt(Ph) from Pyrococcus horikoshi to examine conformational changes in HP2. Surprisingly, the cotransported substrates Na(+) and aspartate induce opposite movements of HP2. We find that in the apo state, HP2 is in a similar conformation as in the aspartate-bound closed state. Na(+) binding to the apo state opens HP2, whereas the subsequent binding of aspartate closes HP2. Our findings show that Na(+) binding opens and stabilizes the extracellular gate, thereby allowing for amino acid substrate binding. In contrast, in the absence of Na(+) and aspartate, HP2 closes, suggesting a potential mechanism for the translocation of the empty binding pocket necessary to complete the transport cycle. The finding that physiological Na(+) concentrations stabilize the open HP2 state would ensure that the outward-facing conformation of the transporter is maintained in physiological solutions and that glutamate transporters are ready to quickly bind glutamate released from glutamatergic synapses.
authors.affiliation: Neuroscience Graduate Program, School of Medicine, Oregon Health and Science University, Beaverton, Oregon 97006-8921, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Focke
authors.forename: Pierre
authors.initials: P
authors.lastname: Moenne-Loccoz
authors.forename: H Peter
authors.initials: HP
authors.lastname: Larsson
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D001692:Biological Transport; D018698:Glutamic Acid / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D043902:Pyrococcus horikoshii; D012964:Sodium / Q000378:metabolism*; D013113:Spin Labels; D013379:Substrate Specificity
chemical_list: D027322:Amino Acid Transport System X-AG; D013113:Spin Labels; D018698:Glutamic Acid; D012964:Sodium