title: Evidence for a third sodium-binding site in glutamate transporters suggests an ion/substrate coupling model.
abstract: Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co- and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, GltPh, only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of GltPh as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in GltPh) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs.
authors.affiliation: Department of Physiology and Biophysics, University of Miami, Miami, FL 33101, USA. Plarsson@med.miami.edu
authors.forename: H Peter
authors.initials: HP
authors.lastname: Larsson
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Wang
authors.forename: Bogdan
authors.initials: B
authors.lastname: Lev
authors.forename: Isabelle
authors.initials: I
authors.lastname: Baconguis
authors.forename: David A
authors.initials: DA
authors.lastname: Caplan
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Vyleta
authors.forename: Hans P
authors.initials: HP
authors.lastname: Koch
authors.forename: Ana
authors.initials: A
authors.lastname: Diez-Sampedro
authors.forename: Sergei Y
authors.initials: SY
authors.lastname: Noskov
mesh_terms: D000818:Animals; D001665:Binding Sites; D002412:Cations / Q000737:chemistry; D003198:Computer Simulation; D050580:Excitatory Amino Acid Transporter 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009010:Monte Carlo Method; D009154:Mutation; D009865:Oocytes; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014982:Xenopus laevis
chemical_list: D002412:Cations; D050580:Excitatory Amino Acid Transporter 3; C493400:SLC1A1 protein, human; D012964:Sodium