title: Permeation and gating residues in serotonin transporter.
abstract: The third transmembrane domain (TM3) of serotonin transporter (SERT) contains two isoleucine residues previously proposed to be involved in binding and transport of serotonin. When Ile-172 was replaced with cysteine, SERT became sensitive to inactivation by externally added [2-(trimethylammonium)ethyl]methanethio-sulfonate (MTSET). The disulfide product of this inactivation was not sensitive to reduction by externally added sulfhydryl compounds, but apparently reacted with intracellular reducing agents to spontaneously regenerate active SERT. The apparent accessibility of this residue to both external and cytoplasmic reagents is consistent with its localization near a serotonin binding site that is alternately exposed to both internal and external media. In another SERT mutant, I179C, transport also was inactivated by MTSET but substrate binding was resistant. External substrate bound to the inactivated I179C and enhanced its reactivation by free thiols. In norepinephrine transporter (NET), cysteine replacement of Ile-155 (corresponding to SERT Ile-179) also rendered the transporter sensitive to MTSET inactivation. In NET I155C, cocaine enhanced this inactivation, and the substrate, dopamine, apparently protected against inactivation. The characteristics of this protection suggest that dopamine was transported, converting NET to a form in which Ile-155 was occluded. The results support the proposal that TM3 of SERT and NET constitute part of the substrate permeation pathway, and that Ile-172 in SERT resides close to the substrate binding site. They also suggest that Ile-179 in SERT (and Ile-155 in NET) is in a conformationally sensitive part of TM3, which may act as part of an external gate.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Chen
authors.forename: G
authors.initials: G
authors.lastname: Rudnick
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003042:Cocaine / Q000494:pharmacology; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D017136:Ion Transport; D007532:Isoleucine / Q000502:physiology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008698:Mesylates / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010084:Oxidation-Reduction; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D027981:Symporters
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; C493227:SLC6A2 protein, human; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; D007532:Isoleucine; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D012701:Serotonin; D006859:Hydrogen; D012964:Sodium; D003042:Cocaine; D011188:Potassium