title: Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter.
abstract: Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.
authors.affiliation: Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600, USA.
authors.forename: E L
authors.initials: EL
authors.lastname: Barker
authors.forename: K R
authors.initials: KR
authors.lastname: Moore
authors.forename: F
authors.initials: F
authors.lastname: Rakhshan
authors.forename: R D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001224:Aspartic Acid; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000032:analysis; D037281:Calnexin; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D026121:Indole Alkaloids; D007425:Intracellular Membranes / Q000737:chemistry; D007457:Iodine Radioisotopes; D015640:Ion Channel Gating / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D004130:N,N-Dimethyltryptamine / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000493:pharmacokinetics*; D050486:Serotonin Plasma Membrane Transport Proteins; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D014162:Transfection; D014316:Tritium; D014981:Xenopus
chemical_list: D018759:Adrenergic Uptake Inhibitors; D000470:Alkaloids; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D026121:Indole Alkaloids; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D017366:Serotonin Receptor Agonists; C493232:Slc6a4 protein, rat; D014316:Tritium; D037281:Calnexin; D001224:Aspartic Acid; D012701:Serotonin; D012965:Sodium Chloride; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; C007884:gramine; D003042:Cocaine; D007099:Imipramine; D004130:N,N-Dimethyltryptamine