Membrane cholesterol regulates inhibition and substrate transport by the glycine transporter, GlyT2

Membrane cholesterol binds to and modulates the function of various SLC6 neurotransmitter transporters, including stabilizing the outward-facing conformation of the dopamine and serotonin transporters. Here, we investigate how cholesterol binds to GlyT2 (SLC6A5), modulates glycine transport rate, and influences bio-active lipid inhibition of GlyT2. Bioactive lipid inhibitors are analgesics that bind to an allosteric site accessible from the extracellular solution when GlyT2 adopts an outward-facing conformation. Using molecular dynamics simulations, mutagen-esis, and cholesterol depletion experiments, we show that bio-active lipid inhibition of glycine transport is modulated by the recruitment of membrane cholesterol to a binding site formed by transmembrane helices 1, 5, and 7. Recruitment involves cho-lesterol flipping from its membrane orientation, and insertion of the 39 hydroxyl group into the cholesterol binding cavity, close to the allosteric site. The synergy between cholesterol and allosteric inhibitors provides a novel mechanism of inhibition and a po-tential avenue for the development of potent GlyT2 inhibitors as alternative therapeutics for the treatment of neuropathic pain and therapeutics that target other SLC6 transporters.