The Formation of a Chloride Channel at the Interface between the Transport and Trimerisation Domains of a Glutamate Transporter

The concentration of glutamate within the glutamatergic synapse is tightly regulated by the excitatory amino acid transporters (EAATs). In addition to their primary role of clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl- conductance. Several crystal structures of an archaeal EAAT homologue, GltPh, at different stages of the transport cycle have been solved. In a recent structure, an aqueous cavity located at the interface of the transport and trimerisation domains has been identified. This cavity is lined by polar residues, several of which have been implicated in Cl− permeation. We hypothesise that throughout the transport cycle this cavity opens to form the Cl− channel. Residues lining this cavity in EAAT1, including Ser-366, Leu-369, Phe-373, Arg-388, Pro-392, and Thr-396 were mutated to small hydrophobic residues. Wild type and mutant transporters were expressed in Xenopus laevis oocytes and two-electrode voltage clamp electrophysiology and radiolabelled substrate uptake were used to investigate function. Significant alterations in substrate-activated Cl- conductance were observed for several mutant transporters. These alterations support the hypothesis that this aqueous cavity at the interface of the transport and trimerisation domains is a partially formed Cl- channel which opens to form a pore through which Cl- ions pass.

Source: BJ Cell - October 20, 2014 Category: Biochemistry Authors: R J Cater, R J Vandenberg, R M Ryan Tags: BJ Cell Source Type: research