No abstract

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity 1 . Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism [2][3][4][5] . Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport [6][7][8] . However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl -) channels, but the physiological role of Clconductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Clchannel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear.Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations resulting in decreased hEAAT1 Clchannel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Clhomeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na + ) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Excitatory amino acid transporters (EAATs) are glutamate transporters that belong to the solute carrier 1A (SLC1A) family. They couple glutamate transport to the cotransport of three sodium (Na+) ions and one proton (H+) and the counter-transport of one potassium (K+) ion. In addition to this coupled transport, binding of cotransported species to EAATs activates a thermodynamically uncoupled chloride (Cl–) conductance. Structures of SLC1A family members have revealed that these transporters use a twisting elevator mechanism of transport, where a mobile transport domain carries substrate and coupled ions across the membrane, while a static scaffold domain anchors the transporter in the membrane. We recently demonstrated that the uncoupled Cl– conductance is activated by the formation of an aqueous pore at the domain interface during the transport cycle in archaeal Glt Ph . However, a pathway for the uncoupled Cl– conductance has not been reported for the EAATs, and it is unclear if such a pathway is conserved. Here, we employ all-atom molecular dynamics (MD) simulations combined with enhanced sampling, free-energy calculations, and experimental mutagenesis to approximate large-scale conformational changes during the transport process and identified a Cl–-conducting conformation in human EAAT1 (hEAAT1). Sampling the large-scale structural transitions in hEAAT1 allowed us to capture an intermediate conformation formed during the transport cycle with a continuous aqueous pore at the domain interface. The free-energy calculations performed for the conduction of Cl– and Na+ ions through the captured conformation highlight the presence of two hydrophobic gates that control low-barrier movement of Cl– through the aqueous pathway. Overall, our findings provide insights into the mechanism by which a human neurotransmitter transporter supports functional duality of active transport and passive Cl– permeation and confirm the commonality of this mechanism in different members of the SLC1A family.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity 1 . Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism 2-5 . Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport 6-8 . However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.