title: Conformational flexibility of transmembrane helix VII of the human serotonin transporter impacts ion dependence and transport.
abstract: The serotonin transporter (SERT) regulates the serotonin concentration in the synapse and is a target of several antidepressant and psychostimulant drugs. Previous work suggested that the middle transmembrane helices (TMHs) of the biogenic amine transporters (TMHs) play a role in substrate and ion recognition. We focused our present studies on exploring the role of TMH VII in transporter function and ion recognition. Residues divergent between human SERT and Drosophila SERT (hSERT and dSERT, respectively) were identified and mutated in hSERT to the corresponding identity in dSERT. hSERT mutants V366S, M370L, S375A, and T381S exhibited a decrease in transport capacity. To further explore the role of these residues in the transport process, we generated cysteine mutants at multiple positions. Pretreatment with [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) caused a decrease in transport of [(3)H]5-HT in the V366C and M370C mutants. The hSERT V366S, M370L, and M370C mutations also altered the sodium and chloride dependence for substrate transport. Interpretation of our results in the context of a homology model of SERT based on the crystal structure of the Aquifex aeolicus leucine transporter suggests flexibility in the conformation of TMH VII that impacts ion dependence and substrate transport.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy and Pharmaceutical Sciences, West Lafayette, IN 47907-2091, United States.
authors.forename: Cody J
authors.initials: CJ
authors.lastname: Wenthur
authors.forename: Gustavo J
authors.initials: GJ
authors.lastname: Rodríguez
authors.forename: Charles P
authors.initials: CP
authors.lastname: Kuntz
authors.forename: Eric L
authors.initials: EL
authors.lastname: Barker
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000493:pharmacokinetics; D001692:Biological Transport; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology; D012964:Sodium / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D002712:Chlorides; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium; D015655:1-Methyl-4-phenylpyridinium