Anion Permeation through Excitatory Amino Acid Transporters

Glutamatergic synaptic transmission critically depends on excitatory amino acid transporters (EAATs) that remove released neurotransmitters from the synaptic cleft and thereby ensure low extracellular glutamate concentrations in the central nervous system. EAATs are thermodynamically coupled glutamate/Na+/H+/K+ transporters and anion-selective channels. EAAT anion channels control neuronal excitability and synaptic communication, and their physiological importance is further corroborated by the recently identified association of altered EAAT anion conduction with neurological disorders. The five mammalian EAATs differ in their effectiveness as glutamate transporters and anion channels. However, pore properties of the known isoforms such as anion selectivity and unitary current amplitudes appear to be closely similar. Although important structural information on secondary-active glutamate transport has been resolved in recent years, the molecular mechanisms underlying anion permeation are still unknown. We here performed molecular dynamics (MD) simulations of the prokaryotic EAAT homologue GltPh to elucidate how these transporters conduct anions. Our results are validated by fluorescence quenching experiments on single-tryptophane mutants of GltPh and patch-clamp recordings of mammalian EAATs. Whereas outward- and inward-facing conformations of GltPh were found to be non-conductive in MD simulations, a voltage-dependent lateral movement of the mobile glutamate transport domain from an intermediate conformation led to the opening of an anion-selective conduction pathway. Amino acid substitutions of homologous pore-forming residues have similar effects on experimental EAAT2/EAAT4 and simulated GltPh single-channel conductances and anion/cation selectivities. Thus, the here identified anion conduction pathway appears to be conserved within the whole glutamate transporter family. Our results highlight how the glutamate transporter family accommodates an anion channel together with a transporter in one single protein.