Oral Presentation 51st Lorne Proteins Conference 2026

One high-order ion coupling mechanism generates multiple SLC1A stoichiometries (132765)

Yan Jiang 1 , Qianyi Wu 1 2 , Chelsea Briot 1 , Ashkan Fakharzadeh Ghaan 3 , Hariprasad Venugopal 4 , Emad Tajkhorshid 3 , Renae Ryan 1
  1. Transporter Biology Group, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, Australia
  2. Department of Biochemistry & Biophysics, Weill Cornell Medicine, 1300 York Ave, New York, NY, USA
  3. Theoretical and Computational Biophysics Group, NIH Resource for Macromolecular Modeling and Visualization, Beckman Institute for Advanced Science and Technology, Department of Biochemistry, and Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA
  4. Ramaciotti Centre for Cryo Electron Microscopy, Monash University, Clayton, VIC, Australia

Precise regulation of extracellular glutamate is critical for maintaining synaptic transmission and preventing excitotoxicity. In mammals, Excitatory Amino Acid Transporters (EAATs) terminate excitatory neurotransmission by transporting glutamate into glial cells and neurons through an electrogenic process coupled to the co-transport of Na+ and H+ and counter-transport of K+1,2. This tightly coupled transport cycle also activates an uncoupled Cl– conductance whose physiological role remains unclear. Despite extensive study, the molecular basis of this complex ion coupling and the diversity of substrate specificity among EAAT family members remains incompletely understood.

Here, we leverage the Drosophila melanogaster EAAT2 (dEAAT2) transporter as a tractable model to dissect the evolution and mechanism of ion coupling in this family. Unlike mammalian homologues, dEAAT2 also transports the osmolyte taurine3. We show that dEAAT2 functions as an electroneutral amino acid exchanger, activating a Cl– conductance that is independent of cation transport. Interestingly, taurine uptake requires Cl– binding, revealing a unique Cl–-dependent substrate recognition mechanism not observed in other SLC1 transporters.

Using cryo-electron microscopy, we determined high-resolution structures of dEAAT2 bound with aspartate or taurine. These structures reveal that a bound Cl– ion occupies the position of the second carboxyl group on aspartate, enabling taurine accommodation within the substrate-binding pocket. Our molecular simulations further show that K+ dependence in human EAATs arises from the coordinated movements of Na+ and H+ binding sites, providing a mechanistic link between these coupled ions.

Remarkably, by introducing two key point mutations, we reconstituted human-EAAT-like electrogenic coupling in dEAAT2, converting its neutral exchange mechanism into Na+/H+/K+-dependent transport. Together, these findings illustrate how EAAT family members have diversified ion-coupling and substrate recognition strategies to fulfil distinct physiological roles while maintaining a conserved molecular framework for complex transport energetics.

Reference

  1. Vandenberg RJ, & Ryan RM (2013). Mechanisms of glutamate transport. Physiological Reviews 93: 1621–1657.
  2. Zerangue N, & Kavanaugh MP (1996). Flux coupling in a neuronal glutamate transporter. Nature 383: 634–637.
  3. Besson MT, Ré DB, Moulin M, & Birman S (2005). High affinity transport of taurine by the Drosophila aspartate transporter dEAAT2. Journal of Biological Chemistry 280: 6621–6626.