The Interface between the Transport and the Trimerisation Domains of Glutamate Transporters Forms a Chloride Channel — ASN Events
  1. Schedule
  2. Session 5: Poster Session A
  3. The Interface between the Transport and the Trimerisation Domains of Glutamate Transporters Forms a Chloride Channel

The Interface between the Transport and the Trimerisation Domains of Glutamate Transporters Forms a Chloride Channel (#101)

Rosemary J Cater 1 , Robert J Vandenberg 1 , Renae M Ryan 1
  1. Discipline of Pharmacology, Bosch Institute, School of Medical Sciences, University of Sydney, Sydney, NSW, Australia

The concentration of glutamate within a glutamatergic synapse is tightly regulated by the excitatory amino acid transporters (EAATs). In addition to their primary role of clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl- conductance. This Cl- conductance is activated by the binding of substrate and Na+, and the direction of Cl- flux is independent of the rate or direction of substrate transport, and thus the two processes are thermodynamically uncoupled1 2 3 . This Cl- conductance is present in all members of the glutamate transporter family and has been proposed to play roles in regulating ionic homeostasis and glutamate release in the retina4 . Several crystal structures of an EAAT archaeal homologue, GltPh, at different stages of the transport cycle have been solved5 6 7 8 . In a recent structure8 , a small cavity located at the interface of the transport and trimerisation domains has been identified and is lined by polarisable residues, several of which have been implicated in Cl− permeation9 10 11 . We hypothesize that throughout the transport cycle this cavity opens up to form the Cl− channel. Residues which line this cavity in EAAT1, including Ser366, Leu369, Phe373, Arg388, Pro392, and Thr396 were mutated to small hydrophobic residues. Wild type and mutant transporters were expressed in Xenopus laevis oocytes and two-electrode voltage clamp electrophysiology and radiolabelled substrate uptake were used to investigate function. Significant alterations in substrate-activated Cl- conductance properties were observed for several mutant transporters while no variations in substrate transport properties were observed. These results support the hypothesis that this aqueous cavity at the interface of the transport and trimerisation domain is a partially formed Cl- channel which mediates chloride permeation through members of the glutamate transporter family.            

  1. Fairman, W. A., Vandenberg, R. J., Arriza, J. L., Kavanaugh, M. P., and Amara, S. G. (1995) An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature 375, 599-603
  2. Wadiche, J. I., Amara, S. G., and Kavanaugh, M. P. (1995) Ion fluxes associated with excitatory amino acid transport. Neuron 15, 721-728
  3. Zerangue, N., and Kavanaugh, M. P. (1996) Flux coupling in a neuronal glutamate transporter. Nature 383, 634-637
  4. Veruki, M. L., Morkve, S. H., and Hartveit, E. (2006) Activation of a presynaptic glutamate transporter regulates synaptic transmission through electrical signaling. Nat Neurosci 9, 1388-1396
  5. Yernool, D., Boudker, O., Jin, Y., and Gouaux, E. (2004) Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431, 811-818
  6. Boudker, O., Ryan, R. M., Yernool, D., Shimamoto, K., and Gouaux, E. (2007) Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445, 387-393
  7. Reyes, N., Ginter, C., and Boudker, O. (2009) Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462, 880-885
  8. Verdon, G., and Boudker, O. (2012) Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog. Nat Struct Mol Biol 19, 355-357
  9. Ryan, R. M., Mitrovic, A. D., and Vandenberg, R. J. (2004) The chloride permeation pathway of a glutamate transporter and its proximity to the glutamate translocation pathway. J Biol Chem 279, 20742-20751
  10. Huang, S., and Vandenberg, R. J. (2007) Mutations in transmembrane domains 5 and 7 of the human excitatory amino acid transporter 1 affect the substrate-activated anion channel. Biochemistry 46, 9685-9692
  11. Kovermann, P., Machtens, J. P., Ewers, D., and Fahlke, C. (2010) A conserved aspartate determines pore properties of anion channels associated with excitatory amino acid transporter 4 (EAAT4). J Biol Chem 285, 23676-23686