title: Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.
abstract: Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+) ions, one H(+) and the counter-transport of one K(+) ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph), whose three dimensional structure is known is also coupled to three Na(+) ions but only two Na(+) ion binding sites have been observed in the crystal structure of Glt(Ph). In order to fully utilize the Glt(Ph) structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph) and accurately determine the number and location of Na(+) ions coupled to transport. Several sites have been proposed for the binding of a third Na(+) ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph) and reveal a new site for the third Na(+) ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph), and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+) compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+) ion in Glt(Ph) and EAAT1.
authors.affiliation: School of Physics, The University of Sydney, Sydney, Australia.
authors.forename: Turgut
authors.initials: T
authors.lastname: Bastug
authors.forename: Germano
authors.initials: G
authors.lastname: Heinzelmann
authors.forename: Serdar
authors.initials: S
authors.lastname: Kuyucak
authors.forename: Marietta
authors.initials: M
authors.lastname: Salim
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Vandenberg
authors.forename: Renae M
authors.initials: RM
authors.lastname: Ryan
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics*; D027341:Excitatory Amino Acid Transporter 1 / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D012964:Sodium / Q000378:metabolism*
chemical_list: D027322:Amino Acid Transport System X-AG; D027341:Excitatory Amino Acid Transporter 1; C493394:SLC1A3 protein, human; D012964:Sodium