Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2

• Published in: PloS one Vol. 11; no. 6; p. e0157583
• Main Authors: Subramanian, Nandhitha, Scopelitti, Amanda J, Carland, Jane E, Ryan, Renae M, O'Mara, Megan L, Vandenberg, Robert J
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 23.06.2016; Public Library of Science (PLoS)
• Subjects: Animals; Betaine; Binding Sites; Bioinformatics; Biology and Life Sciences; Chloride; Computational chemistry; Computer simulation; Coupling (molecular); Crystal structure; Crystallization; Dopamine; Dopamine transporter; Drosophila; Fruit flies; Glycine; Glycine Plasma Membrane Transport Proteins - chemistry; Glycine Plasma Membrane Transport Proteins - genetics; Glycine Plasma Membrane Transport Proteins - metabolism; Glycine transporter; Ion flux; Ions; Leucine; Medicine and Health Sciences; Models, Molecular; Molecular biology; Molecular dynamics; Molecular Dynamics Simulation; Mutation; Pharmacology; Physical Sciences; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Research and Analysis Methods; Sequence Homology, Amino Acid; Simulation; Sodium - metabolism; Synaptic transmission; Transport; Xenopus laevis; Australia; United States > US; Canberra Australian Capital Territory Australia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0157583

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.