title: Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.
abstract: The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.
authors.affiliation: Research School of Chemistry, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Nandhitha
authors.initials: N
authors.lastname: Subramanian
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, University of Sydney, Sydney, NSW, 2006, Australia.|Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, 10021, United States of America.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Scopelitti
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Carland
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Renae M
authors.initials: RM
authors.lastname: Ryan
authors.affiliation: Research School of Chemistry, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Megan L
authors.initials: ML
authors.lastname: O'Mara
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, University of Sydney, Sydney, NSW, 2006, Australia.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Vandenberg
mesh_terms: D000818:Animals; D001665:Binding Sites; D050487:Glycine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; D011994:Recombinant Proteins; D012964:Sodium