American Chemical Society
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Microscopic Characterization of the Chloride Permeation Pathway in the Human Excitatory Amino Acid Transporter 1 (EAAT1)

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posted on 2022-02-22, 19:00 authored by Shashank Pant, Qianyi Wu, Renae Ryan, Emad Tajkhorshid
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.