PT  - JOURNAL ARTICLE
AU  - Zachary J Frangos
AU  - Katie A Wilson
AU  - Heather M Aitken
AU  - Ryan Cantwell Chater
AU  - Robert J Vandenberg
AU  - Megan L O’Mara
TI  - Membrane cholesterol regulates inhibition and substrate transport by the glycine transporter, GlyT2
AID  - 10.26508/lsa.202201708
DP  - 2023 Apr 01
TA  - Life Science Alliance
PG  - e202201708
VI  - 6
IP  - 4
4099  - https://www.life-science-alliance.org/content/6/4/e202201708.short
4100  - https://www.life-science-alliance.org/content/6/4/e202201708.full
SO  - Life Sci. Alliance2023 Apr 01; 6
AB  - 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 bioactive 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, mutagenesis, and cholesterol depletion experiments, we show that bioactive 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 cholesterol flipping from its membrane orientation, and insertion of the 3′ 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 potential avenue for the development of potent GlyT2 inhibitors as alternative therapeutics for the treatment of neuropathic pain and therapeutics that target other SLC6 transporters.