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  • br Funding This work was supported by

    2021-11-20


    Funding This work was supported by grants of the National Academy of Sciences of Ukraine within the programs “Molecular and cellular biotechnologies for medicine, industry and agriculture” (#35-2018), “Scientific Space Research for 2018-2022” (#19-2018), “Smart sensory devices of a new generation based on modern materials and technologies for 2018–2022” (#18-2018) and by State Fund for Fundamental Research of Ukraine (#F76/13-2018). This work was also supported by Cedars Sinai Medical Center’s International Research and Innovation Management Program, the Association for Regional Cooperation in the Fields of Health, Science and Technology, and the participating Cedars-Sinai Medical Center and by the President of the Association Dr. S. Vari.
    Competing financial interest statement
    Author contributions
    Acknowledgements
    Introduction Secondary active glutamate transport by excitatory amino GFP Quantitation Kit transporters (EAATs) (Kanner and Sharon, 1978) terminates glutamatergic synaptic transmission and regulates glutamate concentrations within the CNS. EAATs can also function as anion-selective channels (Fairman et al., 1995, Wadiche and Kavanaugh, 1998), with EAAT anion channels GFP Quantitation Kit regulating cell excitability and synaptic transmission (Picaud et al., 1995). Their physiological relevance is emphasized by the recent discovery that altered EAAT anion conduction is associated with episodic ataxia and epilepsy (Winter et al., 2012). EAAT anion permeation occurs through a defined anion-selective conduction pathway (Kovermann et al., 2010), which is opened and closed through conformational changes coupled to transitions within the glutamate uptake cycle (Bergles et al., 2002, Machtens et al., 2011a, Otis and Kavanaugh, 2000). The channels are perfectly anion selective (Wadiche and Kavanaugh, 1998) and exhibit unitary current amplitudes, which are small but of a similar size range to those of specialized anion channels (Schneider et al., 2014). The five mammalian EAATs differ in their relative glutamate transport rates and anion currents, resulting in isoform-specific differentiation into efficient transporters associated with small macroscopic anion currents and low-capacity transporters that predominantly conduct anions (Mim et al., 2005). However, the functional properties of the underlying anion channels are very similar for each type (Schneider et al., 2014, Torres-Salazar and Fahlke, 2007), indicating conservation of the anion-conducting pore among functionally specialized transporters. So far, the localization of this conduction pathway, the underlying conformation of the transporter, and the mechanisms of anion permeation have not been identified. We used molecular dynamics (MD) simulations to identify which conformations of the archeal glutamate transporter homolog GltPh (Yernool et al., 2004) permit anion permeation and to characterize the molecular features of anion conduction. We analyzed the conformational changes leading to the formation of an anion-selective pore and observed ion permeation along this path in simulations. Using mutagenesis, fluorescence spectroscopy experiments on GltPh and patch-clamp recordings on mammalian EAATs, we confirmed that the anion channel conformation we identified exists under experimental conditions and that this permeation pathway is utilized by both prokaryotic and mammalian glutamate transporters.
    Results
    Discussion EAAT glutamate transporters are prototypical dual function proteins that operate as both secondary active transporters and anion-selective ion channels. Whereas the key structural features of secondary active glutamate transport have been established (Akyuz et al., 2013, Crisman et al., 2009, Reyes et al., 2009, Shrivastava et al., 2008), structural and mechanistic details of anion permeation have been hitherto unknown. In this study, we used a combination of computational and experimental approaches to determine how this class of transporters mediates anion permeation through an aqueous conduction pathway. MD simulations identified an open channel conformation of GltPh that was consistently formed from various ICs by the lateral movement of the transport domain (Figure 1). Opening of the interface between the transport and trimerization domains is followed by voltage-promoted water entry (Figure 2) and the formation of an anion-selective conduction pathway (Figure 3). We verified the predictions of our simulations by fluorescence spectroscopy and functional studies using mutant transporters. Fluorescence quenching experiments demonstrated that tryptophan residues substituted at positions that project into the predicted conduction pathway come into close contact with permeating anions (Figure 4). Moreover, substitution of pore-forming residues had comparable experimental effects on the two key characteristics of an anion-selective conduction pathway, i.e., anion/cation selectivity and ion permeation rates, as predicted by simulations (Figures 5, 6, and 7). These data indicate that pore-forming residues identified through simulations are indeed the major determinants of anion permeation and selectivity in both GltPh and EAATs. Moreover, they demonstrate that this anion conduction pathway is conserved throughout the glutamate transporter family. Our data thus clarify how a class of secondary active transporters can function as anion-selective channels that are gated by transitions in the transport cycle.