Subramanian N et al. (2016), Identification of a 3rd Na+ Binding Site of the...

XB-ART-53839

PLoS One 2016 Jan 01;116:e0157583. doi: 10.1371/journal.pone.0157583.

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

Subramanian N , Scopelitti AJ , Carland JE , Ryan RM , O'Mara ML , Vandenberg RJ .

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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.

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Species referenced: Xenopus laevis
Genes referenced: slc6a5 slc6a9 tpm3

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	Fig 1. Comparison of the Na binding sites in the LeuTAa (dark grey) and dDaT (light grey) crystal structures and the GlyT2 model (colored helices).Crystallographic Na+ from the LeuTAa Na1 and Na2 sites are shown as dark grey spheres. The three modeled Na+ in GlyT2 are colored purple. Na+ occupies the Na1 and Na2 sites, and the proposed Na’ site of GlYT2. The substrate glycine is shown in CPK spacefill.
	Fig 2. GlyT2 model stability and validation.(A) Backbone RMSD of GlyT2 model over 50 ns for all 5 runs. (B) and (C) shows the Na1 and Na2 binding sites in the GlyT2 model. GlyT2 is shown in a cartoon representation. The residues that bind Na+ (purple spacefill) are shown in licorice representation.
	Fig 3. Initial conformation of the membrane-embedded, equilibrated GlyT2 model in MD simulations.(A) Outward occluded conformation of the GlyT2 model for MD simulation. (B) Water mediated salt bridges between R216 (TM1b) and D633 (TM10) and R216 forms a cation-π interaction with F476 (TM6a). (C) R191 from TM1 and D592 from TM8 form a salt bridge.
	Fig 4. Location of the proposed Na3 site in GlyT2.(A) Final conformation of GlyT2 after 50 ns unrestrained MD simulation. Three Na+ ions (purple spacefill) remain stably bound to GlyT2, occupying the Na1 and Na2 sites, and a third site, Na3, where E648 (CPK spacefill) interacts electrostatically with the Na+ ion. The substrate glycine is shown in CPK spacefill and the membrane headgroups are in licorice representation. (B) A close-up view of the Na3 site. The residues that form the Na3 site are shown in CPK.
	Fig 5. Electrostatic potential of the GlyT2 model.The electrostatic potential, contoured at +2 and -2 kT/e, was calculated at pH 7.0 using 150 mM NaCl and a relative dielectric permittivity of 78.5. Negative potential is red, positive is blue. (A) Electrostatic potential map of the intracellular surface of GlyT2, viewed from the intracellular side, normal to the membrane. Membrane phospholipids are shown as CPK colored lines. The initial placement of the third Na+ is in yellow. The final position of Na+ from MD simulations, bound to the proposed Na3 site, is in green. (B) Side view of the GlyT2 model, from the plane of the membrane, shows the density of the electric field lines. The protein is in gray cartoon representation and the substrate glycine is in CPK spacefill representation. Na+ ions occupying the Na1 and Na2 sites are purple.
	Fig 6. Glycine (top panels) and Na+ (bottom panel) concentration-dependent transport currents mediated by WT GlyT2 and GlyT1 and the Na3 site mutant in GlyT2, E648M mutant.Glycine concentration-dependent transport currents were measured in ND96 and Na+ concentration-dependent transport currents were measured in the presence of an EC90 concentration of glycine for the respective transporters. Currents were normalized to the maximal transport current in each case except for the Na+ concentration dependent currents for the E648M mutant because the currents did not appear to saturate at a maximal Na+ concentration.

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