Structures and membrane interactions of native serotonin transporter in complexes with psychostimulants

The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and illicit psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, the oligomerization state of native SERT (nSERT) and its interactions with potential proteins remain unresolved. Here we develop methods to isolate nSERT from porcine brain, utilize fluorescence-detection size-exclusion chromatography to investigate the nSERT oligomerization state, and report single-particle cryo-electron microscopy structures of nSERT in complexes with methamphetamine and cocaine, providing structural insights into psychostimulant recognition and accompanying nSERT conformations. Methamphetamine and cocaine both bind to SERT central site, stabilizing the transporter in outward and outward-occluded conformations, respectively. We also identify densities attributable to multiple cholesterol molecules, as well as to a potential polyunsaturated lipid bound to SERT allosteric site. Our study establishes that nSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol and lipid molecules.