Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA115

Poster Communications

Sodium-induced conformational changes in a glutamate transporter homologue

C. Alleva1, C. Fahlke1, J. Machtens1

1. Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Aachen, NRW, Germany.

Excitatory amino acid transporters (EAATs) ensure low synaptic glutamate concentration by secondary active transport of the released neurotransmitter back into neurons and glial cells. Glutamate uptake is coupled to the cotransport of three Na+ ions, one proton and the countertransport of one K+ ion; this coupling stoichiometry provides the driving force needed to maintain up to 106-fold glutamate gradients across the membrane, essential to guarantee reliable signal transduction in excitatory synapses. The transport cycle is characterized by two conformational rearrangements: closure of a gate shields the binding pocket from the bulk solution, while an elevator-like transmembrane translocation ensures alternating accessibility of the substrate binding domain to either side of the membrane. Additional regulation is provided by Na+ ion binding: two Na+ ions associate before the substrate and are correlated with the opening of the extracellular gate; binding of the substrate, followed by the third Na+, induces gate closure, which in turn enables translocation across the membrane; the K+ ion then associates from the intracellular side to permit re-translation of the transport domain. Here, we use all-atom molecular dynamics simulations and stopped-flow fluorescence spectroscopy experiments to characterize the coupling between Na+ binding and gate dynamics. We show that Na+ binding is preceded by two events: opening of the extracellular gate and reorganization of the binding pocket. Both are rare events in the apo state, but association of Na+ stabilizes the open, bound-like configuration primed for substrate binding; the coupling between Na+ binding and gate opening thus follows a conformational selection mechanism. In support of these findings, we experimentally demonstrate that two mutants, previously characterized as either affecting sodium coupling or substrate selectivity both exert their action by modifying the extracellular gate equilibrium between open and closed state. Our results illustrate how an allosteric coupling mechanism between Na+ binding, gate dynamics and subsequent substrate binding enables glutamate transporters to fulfil their physiological task by simultaneously accomplishing high glutamate transport rates and high glutamate concentration gradients.

Where applicable, experiments conform with Society ethical requirements