Identification of a 3rd Na+ binding site of the glycine transporter, GlyT2

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

Adapting relationships with place: Investigating the evolving place attachment and ‘sense of place’ of UK higher education students during a period of intense transition

In recent years interest has emerged regarding the geographies of higher education students, particularly in patterns of mobility and dispersion. While anecdotal rhetoric suggests a ‘typical student’ exists within UK institutions. What resonates is the notion that students are inherently heterogeneous, experiencing University in differing ways and times according to their circumstances and year of study. This paper uses ‘walking interviews’ conducted with University of Portsmouth students as a method to unpack how ‘non-local’ students might go about interpreting their sense of place within their term-time location. This methodology was designed specifically to ensure discussions of ‘sense of place’ remain directly in the context of the city and recognises the adaptive relationships students have with their term-time locations. This is important as there is a tendency within the literature to focus solely on the transition into University, ignoring that students often experience pressures throughout their degree pathway. These pressures can be linked to various social and spatial changes, such as insecurities regarding fitting in amongst unfamiliar peer groups or a lack of confidence concerning engagement with academic and non-academic practices, and draws attention to the non-linearity of students’ associations with their term-time location

Rational Design of Highly Cytotoxic η6-Arene β-Diketiminato-Ruthenium Complexes

A series of ruthenium-benzene complexes with β-diketiminate ligands modified with electron-withdrawing groups were prepd. and characterized by NMR spectroscopy, mass spectrometry, and single-crystal x-ray diffraction. The complexes are stable in air and undergo controlled hydrolysis in water. The complexes were evaluated for anticancer activity in vitro, and two of them proved to be highly cytotoxic, comparable or even superior to cisplatin. This work shows the potential utility of the β-diketiminate ligand in the rational design of new anticancer metal-contg. drugs. A related complex with a η6-C6H5CF3 ligand was prepd. and found to undergo a nucleophilic addn. reaction at the coordinated arene ring to afford a substituted η5-cyclohexadienyl deriv

Identification of a 3^rdNa⁺binding site of the glycine transporter, GlyT2

© 2016 Subramanian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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

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

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

Initial conformation of the membrane-embedded, equilibrated GlyT2 model in MD simulations.

<p>(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.</p

Location of the proposed Na3 site in GlyT2.

<p>(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.</p

Comparison of the Na binding sites in the LeuT_Aa (dark grey) and dDaT (light grey) crystal structures and the GlyT2 model (colored helices).

<p>Crystallographic Na⁺ from the LeuT_Aa 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.</p