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.