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. 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. Glutamate transporters, known as Excitatory Amino Acid Transporters (EAATs), also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport. However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown.
I will describe 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 functions and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family. Furthermore, this work assists in understanding the functional roles the chloride channel plays, notably, in maintaining cell excitability and osmotic balance and provides a framework for the rational development of therapeutics that can differentially modulate substrate transport or channel properties for the treatment of neurological disorders caused by EAAT dysfunction such as Episodic Ataxia.