Open AccessIdentification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2
Author(s) -
Nandhitha Subramanian,
Amanda J. Scopelitti,
Jane E. Carland,
Renae M. Ryan,
Megan L. O’Mara,
Robert J. Vandenberg
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0157583
Subject(s) - binding site , betaine , transporter , chemistry , biochemistry , gene
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, LeuT Aa , 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 LeuT Aa . 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 LeuT Aa , 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.