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 GltPh. 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.
Keywords: chloride channel; electrophysiology; excitatory amino acid transporter (EAAT); glutamate transporter; molecular dynamics; structural transition.