Mechanisms of Anion Conduction by Coupled Glutamate Transporters

Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na+/H+/K+ transporters but also function as an channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog Glt(Ph) and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward-and inward-facing Glt(Ph) conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on Glt(Ph) simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.