Inhibitory glycinergic neurotransmission plays an important role in regulating excitatory tone in the ascending pain pathway, and its dysfunction has been implicated in chronic pain. The glycine transporter, GlyT2, is expressed on presynaptic inhibitory neurons and is responsible for the rapid removal of glycine from the synapse and for recycling into synaptic vesicles. Inhibitors targeting GlyT2 could therefore increase glycine in the synapse to restore control of nociception
We recently developed a class of N-acyl amino acids that inhibit GlyT2 and are analgesic in rodent models of chronic pain. These bioactive lipids are comprised of an amino acid head group conjugated to long flexible monounsaturated lipid tail. Importantly, these inhibitors display varying degrees of partial inhibition, which would allow refilling of vesicles. In order to understand how these bioactive lipids inhibit GlyT2, we used mutagenesis with ligand docking and molecular dynamics, to show that they bind to an extracellular allosteric site on the transporter formed by transmembrane helices TM5, TM7, TM8, and the extracellular loop, EL4. Following 100 ns of simulation, they penetrate these TM helices tail first, forging a deep cavity, with the head group stabilised by aromatic residues in TM7, TM8, and EL4 and the tail wedged between aliphatic rich regions of TM5 and TM8. Mutation of F428 (TM5), Y550 (EL4), P561 (TM8), W563 (TM8), and L569 (TM8) produce transporters that are no longer sensitive to inhibition suggesting these residues are critical for stabilising the binding site.
Conversely, “inactive” acyl amino acids that do not cause inhibition of GlyT2 have unstable interactions with GlyT2, and are unable to penetrate transmembrane helices during the simulation. Additionally, inhibitors with shorter tail lengths show reduced potency at GlyT2, and burrow more shallowly in this cavity. We therefore propose that the formation of this deep binding pocket is critical for inhibition. The presence of a lipid tail between TM5, TM7, and TM8 may therefore inhibit GlyT2 by acting as a physical “spanner in the works”, gluing transmembrane helices together to slow, but not prevent transport, which would explain how bioactive lipids are able to act as partial inhibitors.