One high-order ion-coupling mechanism generates multiple SLC1A stoichiometries

Human excitatory amino acid transporter 2 (hEAAT2) is a glial transporter that clears glutamate and aspartate from the extracellular space to terminate neurotransmission. When this essential role is disrupted, excess extracellular glutamate causes excitotoxicity and neurological disease. hEAAT2 transports amino acids into cells against their concentration gradients by coupling uptake to the co-transport of Na + and H + , and counter-transport of K + . Substrate transport also activates a thermodynamically uncoupled Cl - conductance, whose significance is unknown. Here, to dissect the molecular mechanisms underlying coupled and uncoupled ion transport by EAATs, we leverage the tractable Drosophila melanogaster homologue dEAAT2, which transports taurine as well as glutamate and aspartate. By functionally characterising dEAAT2, we confirmed it has features of a canonical EAAT transport cycle, including an uncoupled Cl - conductance. However, it functions an ion-independent electroneutral exchanger, in stark contrast to its ion-coupled electrogenic human counterpart. Cryo-electron microscopy (cryo-EM) and computational simulations confirmed the inability of dEAAT2 to transport cations, but just two point mutations conferred Na + , H + , and K + dependence, reconstituting the complex ion coupling profile of human transporters in the Drosophila exchanger. We also showed that dEAAT2 transports taurine in a complex with Cl - , bestowing a unique Cl - -dependence on taurine transport, independent of uncoupled Cl - flux. Our findings show that ancestral exchangers in the EAAT family preserve the machinery to achieve complex ion-coupled transport, revealing the molecular basis of high-order ion coupling in hEAATs.