Pharmacological modulation of the glutamate transporter chloride channel

Abstract

Excitatory amino acid transporters (EAATs) of the solute carrier 1 (SLC1A) family facilitate glutamate clearance in the central nervous system. The EAATs couple substrate transport to Na+, K+, and H+ electrochemical gradients, rapidly transporting glutamate into surrounding astrocytes and neurons. In addition to the secondary active transport of glutamate, the EAATs mediate a thermodynamically uncoupled Cl− conductance, exhibiting dual function as glutamate transporters and Cl− channels. The SLC1A transporters translocate substrates across the membrane via a twisting elevator mechanism. The cryo-EM structure of an archaeal homologue GltPh in the Cl− conducting state (ClCS) further reveals that the Cl− permeation pathway opens as an intermediate state of the transport cycle. The SLC1A transporters assemble into trimers; whilst individual protomers are largely considered to function independently, anisotropic network model analyses predicted collective motion within the transporter trimer. Functional evidence suggests that large-scale lateral motions may further promote entry into the ClCS. The first aim of this thesis is to capture the proposed collective motion in SLC1A transporter trimers using an interprotomeric cysteine crosslinking strategy. The structure of two GltPh sing-cysteine mutants were determined using cryo-EM. GltPh-I361C adopted an uncrosslinked outward facing state, whilst GltPh-A391C was captured in an uncrosslinked but novel intermediate state, which closely precedes Cl− channel opening. The uncoupled Cl− conductance in EAAT1 contributes to ionic balance across glial and neuronal membranes. The second aim of this thesis is to identify and characterise functionally selective inhibitors of the Cl− channel in EAAT1 as pharmacological tools. The ClCS structure provided a basis of the virtual screening of compounds that targets this state, thereby allowing selective modulation the Cl− conductance without disrupting normal transport function.