Unraveling the molecular mechanisms of transporters from the SLC1A family and elucidating links to dysfunction in brain disease

Abstract

The Solute Carrier 1A (SLC1A) family comprises a group of membrane proteins that act as dual-function amino acid transporters and Cl- channels. It includes the human glutamate transporters, known as the excitatory amino acid transporters (EAATs), and the alanine serine cysteine transporters (ASCTs). The EAATs are glutamate transporters vital in the control of synaptic glutamate concentrations throughout the brain and key to the prevention of excitotoxicity which manifests in neurological malfunction. ASCT1 and 2 are both neutral amino acid exchangers which have been implicated in neurological disease and in the case of ASCT2, cancer.

The objective of this thesis was to elucidate the molecular mechanisms of the SLC1A transporters which dually drive their physiological roles and their involvement in disease. I firstly examined a glutamine transporter inhibitor, L-γ-glutamyl-p-nitroanilide (GPNA), which has been used to evaluate the potential of ASCT2 as an anticancer target. This work revealed a novel mechanism whereby GPNA activates a Cl- conductance in ASCT2 to the same extent as a transported substrate whilst not undergoing the full transport cycle. I also show that in addition to its currently known targets, GPNA inhibits several of the glutamate transporters. Together, these findings raise questions surrounding the true mechanisms of the anticancer effects of GPNA. I also investigated a group of mutations in human glutamate transporter 1 (EAAT1) linked to a rare neurological disorder, episodic ataxia type 6, and provide evidence that it is disturbances to the EAAT1 Cl- conductance which underlies this pathophysiology. Lastly, I uncover the intricate ion-coupling mechanisms of the glutamate transporters using a unique functional homologue, Drosophila EAAT2, showing that Na+, K+ and H+ coupling are all tightly related. Ultimately, the work presented in this thesis will help to propel more effective drug discovery strategies for the SLC1A transporter family.