Exploring the elevator mechanism of the glutamate transporter family: link to function and disease

Abstract

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system, the extracellular concentration of which must be tightly regulated to avoid neurotoxicity. This role is carried out by the Excitatory Amino Acid Transporters (EAATs). The transport of a glutamate molecule via the EAATs is thermodynamically coupled to the cotransport of three Na+ ions, one H+ ion and the counter-transport of one K+ ion. In addition to their primary role of clearing glutamate from the synaptic cleft to terminate glutaminergic transmission, the EAATs also possess a thermodynamically uncoupled Cl- conductance. This dual function is mediated by distinct conformational states of the transporter, and the physiological roles of the Cl- channel include maintaining ionic and osmotic balance, and regulating cell excitability. Dysfunction of the EAATs has been implicated in disease states, including episodic ataxia, glioma, depression, amyotrophic lateral sclerosis, cerebral ischemia, and Alzheimer's disease. The EAATs use the 'twisting elevator' mechanism for substrate transport, and I hypothesise that the Cl- channel is activated at the domain interface during this elevator-like movement. In Chapter 3 of this thesis, I was able to trap the mammalian EAAT1 transporter (hEAAT1) in an outward-facing state (OFS), an inward-facing state (IFS) and in a novel Cl- conducting state (ClCS) using a cysteine cross-linking strategy. X-ray crystallography and cryo-EM structures of these three states were solved by group members, and together this functional and structural data provide the last piece of the puzzle in the glutamate translocation cycle. At the end of this chapter, I also demonstrate that this Cl- channel is gated by two clusters of hydrophobic residues. Dysfunction of the Cl- channel in hEAAT1 has been implicated in diseases including episodic ataxia (EA), which is a rare genetic disorder caused by inherited channelopathies, clinically characterised by progressive, and recurrent episodes of ataxia. EA type 6 (EA6) was first identified in a young child with early-onset episodic ataxia with a single point mutation in the gene SLC1A3 that encodes hEAAT1. It has been demonstrated that a substantial increase in the Cl- channel function leading to the elevated extrusion of Cl- by this mutant transporter contributes to the pathology of EA6. Recently, several more mutations in SLC1A3 have been identified in EA6 patients. In Chapter 4 of this thesis, I have introduced these mutations into hEAAT1 transporter and characterised them using electrophysiology, radiolabelled glutamate uptake, and confocal microscopy to investigate the effects of these mutations on glutamate transporter activity. Together with in vivo studies in a Drosophila melanogaster model conducted by collaborators, my results suggest disruption of Cl- homeostasis in astrocytes as a result of these mutations results in altered Cl- channel activity that may contribute to the pathology of EA6. Cl- homeostasis has essential roles in cell osmotic balance related to apoptosis, and the interplay between excitatory and inhibitory neurotransmission. Cl- homeostasis in glial cells is maintained by a few key players, including NKCC (Na+-K+-Cl- cotransporter), KCC (K+-Cl- cotransporter), VRAC (volume-regulated anion channels), and the EAATs. It has been suggested that VRAC has a role in astrocyte-neuron communication via mediating the release of both glutamate and taurine, which act on excitatory neurons (NMDA and AMPA receptors) and inhibitory neurons (glycine and GABA receptors), respectively. Taurine is an organic osmolyte that plays a crucial role in cell volume regulation, and has also been implicated in many physiological functions, including neurotransmission and membrane stabilisation. A glutamate transporter isoform from Drosophila melanogaster (dEAAT2) has been demonstrated to transport taurine, in addition to aspartate and glutamate, and has recently been reported to play an important role in hearing and sleep modulation in Drosophila melanogaster. To better understand the function of dEAAT2 and how it is related to other members of the SLC1A family, in Chapter 5 of this thesis, I characterised the mechanism of transport for dEAAT2, and discovered that it is a high-affinity aspartate/taurine exchanger, which displays different coupling stoichiometry between the two substrates and possesses an uncoupled Cl- conductance. My results from this chapter may also provide a foundation for the determination of the mammalian EAATs H+/K+ binding sites, which has been under debate for years. The work presented in this thesis has elucidated the intrinsic link between the elevator mechanism and the substrate activated Cl- conductance in the SLC1 glutamate transporter family, and further characterised the physiological role of this Cl- channel in human disease with further implications on the mechanism of transport and neurotransmission.