title: The two Na+ sites in the human serotonin transporter play distinct roles in the ion coupling and electrogenicity of transport.
abstract: Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na(+), Cl(-), and K(+) gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na(+)-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca(2+) (but not other cations) to functionally replace Na(+) for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca(2+) and Na(+) illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca(2+) promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na(+)-binding sites.
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203 and.
authors.forename: Bruce
authors.initials: B
authors.lastname: Felts
authors.forename: Akula Bala
authors.initials: AB
authors.lastname: Pramod
authors.forename: Walter
authors.initials: W
authors.lastname: Sandtner
authors.forename: Nathan
authors.initials: N
authors.lastname: Burbach
authors.forename: Simon
authors.initials: S
authors.lastname: Bulling
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: L Keith
authors.initials: LK
authors.lastname: Henry
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001693:Biological Transport, Active / Q000502:physiology; D004298:Dopamine / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
chemical_list: C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D001216:Asparagine; D012964:Sodium; D004298:Dopamine
keywords: Electrophysiology; Homology Modeling; Ion Dependence; Monoamine Transporters; SLC6; Serotonin Transporters; Structure-Function; Transporters