Functional and structural characterisation of the glycine transporter 2

Abstract

Modulation of glycinergic neurotransmission is a potential new avenue for the development of clinically viable treatments of neuropathic pain. Inhibition of the glycine transporter 2 has renewed interest with opiranserin (GlyT2 IC50= 120-860 nM), a GlyT2 inhibitor, currently being tested in Phase 3 clinical trials. This compound is based on the scaffold of one of the first GlyT2 inhibitors developed, ORG25543 (GlyT2 IC50= 16 nM). The first aim of this thesis was to characterise the mode of inhibition (orthosteric or allosteric) and determine the binding site of this class of compounds.

Three sites have previously been described as modulatory sites of SLC6 transporters, of which GlyT2 is a member. The substrate site, the vestibule allosteric site and the lipid allosteric site. Mutations in the lipid allosteric site but not the substrate site and vestibule allosteric site resulted in decreased potency of ORG25543. Additionally, these mutations resulted in a faster off-rate of ORG25543, however, it is not well correlated to potency. Cholesterol binds to a distinct but connected site to modulate the off rate of these inhibitors.

The second aim of this study was to purify GlyT2 and optimise conditions for structural determination. Thus far, several SLC6 members have an atomic structure resolved including the serotonin transporter, Drosophila dopamine transporter, GABA transporter 1 and glycine transporter 1 but there is no experimental structure of GlyT2. To achieve this, I expressed several constructs which included enhanced green fluorescent protein (eGFP) as a reporter protein and a soluble fusion protein (lichenase). The final construct involved lichenase fused to a truncated N terminus of GlyT2 and a cleavable eGFP fused to the C terminus. This has enabled the first 2D classes to be determined and provides an avenue for a protein structure to be determined.