Glycine receptors are pentameric ligand-gated ion channels that mediate fast inhibitory neurotransmission throughout the nervous system. They have recently emerged as a target for chronic pain therapies due to their role in nociceptive signalling. N-arachidonyl glycine is an endogenous lipid found abundantly within the spinal cord that has been shown to modulate glycine receptors [1] and produce analgesia in animal models of chronic pain [2], however its mechanism of action remains unknown.
The aim of this study was to identify lipids that act as positive allosteric modulators of glycine receptors and characterise how this modulation occurs. A library of 32 acyl-amino acids was initially screened across human glycine receptors using two-electrode voltage clamp electrophysiology. Lipids containing a glycine head group conjugated to an unsaturated lipid tail cause significant potentiation when the double bonds to exists in the cis-conformation within the central region of the tail. From these structure activity relationships, a second series of lipids containing a benzyl-moiety within the centre of lipid tail were synthesised. This further improved the efficacy of lipid modulation by up to 6-fold and increased the potency of receptor activation by over 10-fold. C18 Ortho-benzyl glycine caused the greatest receptor potentiation – incurring a maximal potentiation of 1500% with an EC50 of 664 nM at the homomeric α1 receptor.
Molecular dynamic simulations of C18 ortho-benzyl glycine at the homomeric α1 receptor were conducted and indicated consistent binding within a cavity between transmembrane domains 1 and 4. The benzyl-moiety was found to pi-stack with a tryptophan residue on TM1 (W239), which faces a proline residue on TM4 (P404). This proline induces a kink in the transmembrane domain that impacts on receptor kinetics [3] which could be mechanistically responsible for the lipid’s modulatory activity. Mutagenesis and cryoEM techniques will be used to further explore this hypothesis.