Poster Presentation 11th Annual Conference of the International Chemical Biology Society 2022

Membrane cholesterol regulates inhibitor sensitivity and substrate transport by the glycine transporter, GlyT2 (#129)

Zachary J Frangos 1 , Katie A Wilson 2 3 , Heather M Aitken 2 4 , Ryan P Cantwell Chater 1 , Robert J Vandenberg 1 , Megan L O'Mara 2 4
  1. Molecular Biomedicine Theme, School of Medical Sciences, University of Sydney, Sydney, NSW, Australia
  2. Research School of Chemistry, College of Science, The Australian National University, Canberra, ACT, Australia
  3. Department of Biochemistry, Memorial University of Newfoundland, St, John's, NL, Canada
  4. Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia

The glycine transporter type 2 (GlyT2, SLC6A5) has attracted considerable attention as a therapeutic target for chronic pain. We have developed potent, allosteric bioactive lipid inhibitors of GlyT2 that are analgesic in animal models of chronic pain. Despite promising in vivo results, clinical progression of these compounds is hindered by potential toxicity caused by complete or irreversible inhibition. Understanding the molecular mechanisms that mediate potency and reversibility, is essential to enhancing the clinical viability of this class of analgesics. Membrane cholesterol has been shown to modulate the functionality and pharmacological sensitivity of the serotonin (SLC6A4) and dopamine (SLC6A3) transporters, with a conserved binding site between transmembrane domains 1, 5 and 7 observed in their respective atomic structures1-3. Therefore, this study aimed to determine the effect of cholesterol on GlyT2 function and its sensitivity to inhibition. Cholesterol depletion was achieved by treating Xenopus laevis oocytes expressing hGlyT2 with methyl-beta-cyclodextrin. Functionally, cholesterol depletion reduced Vmax – consistent with cholesterol stabilising the outward-facing conformation of this transporter family4,5. Regarding inhibitor sensitivity, cholesterol depletion reduced the potency of C18 ω9 L-lysine as well as C18 ω9 L-leucine and enhanced the reversibility of C18 ω9 L-carnitine, C18 ω9 L-leucine and ORG25543, whereas no changes were observed in the activity of C18 ω9 L-tryptophan. Molecular dynamics (MD) simulations predicted that compounds affected by cholesterol depletion insert deeper into their binding pocket, kinking TM5 and allowing spontaneous insertion of membrane cholesterol between TMs 1, 5 and 7. Introduction of residues that would disrupt cholesterol binding in this region via mutagenesis, generated similar results to cholesterol depletion. Furthermore, in MD simulations of these mutants the binding of bioactive lipids and their interaction with cholesterol was altered, suggesting this region is critical to the interaction of membrane cholesterol and GlyT2 inhibitors. Understanding the interaction between cholesterol and GlyT2 inhibitors will aid the rational development of compounds with optimised potency and reversibility that can mitigate potential toxicity.

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